Maize cellulose synthases and uses thereof

ABSTRACT

The invention provides isolated cellulose synthase nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering cellulose synthase levels in plants. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions.

RELATED APPLICATIONS

[0001] This continuation application claims the benefit of U.S. patentapplication No. 60/096,822 filed Aug. 17, 1998, U.S. patent applicationSer. No. 09/371,383, filed Aug. 6, 1999, and U.S. patent applicationSer. No. 09/550,483, filed Apr. 14, 2000 all of which are incorporatedherein by reference.

TECHNICAL FIELD

[0002] The present invention relates generally to plant molecularbiology. More specifically, it relates to nucleic acids and methods formodulating their expression in plants.

BACKGROUND OF THE INVENTION

[0003] Polysaccharides constitute the bulk of the plant cell walls andhave been traditionally classified into three categories: cellulose,hemicellulose, and pectin. Fry, S. C. (1988), The growing plant cellwall: Chemical and metabolic analysis, New York: Longman Scientific &Technical. Whereas cellulose is made at the plasma membrane and directlylaid down into the cell wall, hemicellulosic and pectic polymers arefirst made in the Golgi apparatus and then exported to the cell wall byexocytosis. Ray, P. M., et al., (1976), Ber. Deutsch. Bot. Ges. Bd. 89,121-146. The variety of chemical linkages in the pectic andhemicellulosic polysaccharides indicates that there must be tens ofpolysaccharide synthases in the Golgi apparatus. Darvill et al., (1980).The primary cell walls of flowering plants. In The Plant Cell (N. E.Tolbert, ed.), Vol. 1 in Series: The biochemistry of plants: Acomprehensive treatise, eds. P. K. Stumpf and E. E. Conn (New York:Academic Press), pp. 91-162.

[0004] Even though sugar and polysaccharide compositions of the plantcell walls have been well characterized, very limited progress has beenmade toward identification of the enzymes involved in polysaccharidesformation, the reason being their labile nature and recalcitrance tosolubilization by available detergents. Sporadic claims for theidentification of cellulose synthase from plant sources have been madeover the years. Callaghan, T., and Benziman, M. (1984), Nature 311,165-167; Okuda, et al., (1993), Plant Physiol. 101, 1131-1142. However,these claims have been met with skepticism. Callaghan, T., and Benziman,M. (1985), Nature 314, 383-384; Delmer, et al., (1993), Plant Physiol.103, 307-308. It was only recently that a putative gene for plantcellulose synthase (CeIA) was cloned from the developing cotton fibersbased on homology to the bacterial gene. Pear, et al., Proc. Natl. Acad.Sci. (USA) 93, 12637-12642; Saxena, et al., (1990), Plant MolecularBiology 15, 673-684; see also, WO 9818949.

[0005] Cellulose, by virtue of its ability to form semicrystallinemicrofibrils has a very high tensile strength which approaches that ofsome metals. Niklas, K. J. (1992), Plant Biomechanics: An engineeringapproach to plant form and function, The University of Chicago Press, p.607. Bending strength of the culm of normal and brittle-culm mutants ofbarley has been found to be directly correlated with the concentrationof cellulose in the cell wall. Kokubo, et al., (1989), Plant Physiology91, 876-882; Kokubo, et al., (1991) Plant Physiology 97, 509-514.

[0006] As stalk composition contributes to numerous quality factorsimportant in maize breeding, what is needed in the art are products andmethods for manipulating cellulose concentration in the cell wall andthereby altering plant stalk quality to provide, for example, increasedstandability or improved silage. The present invention provides theseand other advantages.

SUMMARY OF THE INVENTION

[0007] Generally, it is the object of the present invention to providenucleic acids and proteins relating to cellulose synthases. It is anobject of the present invention to provide transgenic plants comprisingthe nucleic acids of the present invention, and methods for modulating,in a transgenic plant, expression of the nucleic acids of the presentinvention.

[0008] Therefore, in one aspect the present invention relates to anisolated nucleic acid comprising a member selected from the groupconsisting of (a) a polynucleotide having a specified sequence identityto a polynucleotide encoding a polypeptide of the present invention; (b)a polynucleotide which is complementary to the polynucleotide of (a);and, (c) a polynucleotide comprising a specified number of contiguousnucleotides from a polynucleotide of (a) or (b). The isolated nucleicacid can be DNA.

[0009] In other aspects the present invention relates to: 1) recombinantexpression cassettes, comprising a nucleic acid of the present inventionoperably linked to a promoter, 2) a host cell into which has beenintroduced the recombinant expression cassette, and 3) a transgenicplant comprising the recombinant expression cassette. The host cell andplant are optionally from maize, wheat, rice, or soybean.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Overview

[0011] A. Nucleic Acids and Protein of the Present Invention

[0012] Unless otherwise stated, the polynucleotide and polypeptidesequences identified in Table 1 represent polynucleotides andpolypeptides of the present invention. Table 1 cross-references thesepolynucleotide and polypeptides to their gene name and internal databaseidentification number. A nucleic acid of the present invention comprisesa polynucleotide of the present invention. A protein of the presentinvention comprises a polypeptide of the present invention.

[0013] Table 2 further provides a calculation of the percentidentity/similarity of the referenced polynucleotide/polypeptidesequences to homologues identified using methods such as the onedisclosed in Example 4. TABLE 1 Polynucleotide Polypeptide SEQ Gene NameDatabase ID NO: SEQ ID NO: ID NO: Cellulose Cdpgs45 (cesA-3) 1 2synthase Cellulose Cqrae19 (cesA-9) 5 6 synthase

[0014] B. Exemplary Utility of the Present Invention

[0015] The present invention provides utility in such exemplaryapplications as improvement of stalk quality for improved stand orsilage. Further, the present invention provides for an increasedconcentration of cellulose in the pericarp, hardening the kernel andthus improving its handling ability.

[0016] Definitions

[0017] Units, prefixes, and symbols may be denoted in their SI acceptedform. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively. Numeric rangesrecited within the specification are inclusive of the numbers definingthe range and include each integer within the defined range. Amino acidsmay be referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBMBNomenclature Commission. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes. Unless otherwise providedfor, software, electrical, and electronics terms as used herein are asdefined in The New IEEE Standard Dictionary of Electrical andElectronics Terms (5^(th) edition, 1993). The terms defined below aremore fully defined by reference to the specification as a whole. Sectionheadings provided throughout the specification are not limitations tothe various objects and embodiments of the present invention.

[0018] By “amplified” is meant the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequences as atemplate. Amplification systems include the polymerase chain reaction(PCR) system, ligase chain reaction (LCR) system, nucleic acid sequencebased amplification (NASBA, Cangene, Mississauga, Ontario), Q-BetaReplicase systems, transcription-based amplification system (TAS), andstrand displacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

[0019] As used herein, “antisense orientation” includes reference to aduplex polynucleotide sequence that is operably linked to a promoter inan orientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

[0020] By “encoding” or “encoded”, with respect to a specified nucleicacid, is meant comprising the information for translation into thespecified protein. A nucleic acid encoding a protein may comprisenon-translated sequences (e.g., introns) within translated regions ofthe nucleic acid, or may lack such intervening non-translated sequences(e.g., as in cDNA). The information by which a protein is encoded isspecified by the use of codons. Typically, the amino acid sequence isencoded by the nucleic acid using the “universal” genetic code. However,variants of the universal code, such as are present in some plant,animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, orthe ciliate Macronucleus, may be used when the nucleic acid is expressedtherein.

[0021] When the nucleic acid is prepared or altered synthetically,advantage can be taken of known codon preferences of the intended hostwhere the nucleic acid is to be expressed. For example, although nucleicacid sequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498(1989)). Thus, the maize preferred codon for a particular amino acid maybe derived from known gene sequences from maize. Maize codon usage for28 genes from maize plants is listed in Table 4 of Murray et al., supra.

[0022] As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of a native (non-synthetic), endogenous, biologically (e.g.,structurally or catalytically) active form of the specified protein.Methods to determine whether a sequence is full-length are well known inthe art, including such exemplary techniques as northern or westernblots, primer extension, S1 protection, and ribonuclease protection.See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Comparison to known full-lengthhomologous (orthologous and/or paralogous) sequences can also be used toidentify full-length sequences of the present invention. Additionally,consensus sequences typically present at the 5′ and 3′ untranslatedregions of mRNA aid in the identification of a polynucleotide asfull-length. For example, the consensus sequence ANNNNAUGG, where theunderlined codon represents the N-terminal methionine, aids indetermining whether the polynucleotide has a complete 5′ end. Consensussequences at the 3′ end, such as polyadenylation sequences, aid indetermining whether the polynucleotide has a complete 3′ end.

[0023] As used herein, “heterologous” in reference to a nucleic acid isa nucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by human intervention. For example, apromoter operably linked to a heterologous structural gene is from aspecies different from that from which the structural gene was derived,or, if from the same species, one or both are substantially modifiedfrom their original form. A heterologous protein may originate from aforeign species or, if from the same species, is substantially modifiedfrom its original form by human intervention.

[0024] By “host cell” is meant a cell which contains a vector andsupports the replication and/or expression of the vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

[0025] The term “introduced” includes reference to the incorporation ofa nucleic acid into a eukaryotic or prokaryotic cell where the nucleicacid may be incorporated into the genome of the cell (e.g., chromosome,plasmid, plastid or mitochondrial DNA), converted into an autonomousreplicon, or transiently expressed (e.g., transfected mRNA). The termincludes such nucleic acid introduction means as “transfection”,“transformation” and “transduction”.

[0026] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with it as found in itsnatural environment. The isolated material optionally comprises materialnot found with the material in its natural environment; or (2) if thematerial is in its natural environment, the material has beensynthetically altered or synthetically produced by deliberate humanintervention and/or placed at a different location within the cell. Thesynthetic alteration or creation of the material can be performed on thematerial within or apart from its natural state. For example, anaturally-occurring nucleic acid becomes an isolated nucleic acid if itis altered or produced by non-natural, synthetic methods, or if it istranscribed from DNA which has been altered or produced by non-natural,synthetic methods. The isolated nucleic acid may also be produced by thesynthetic re-arrangement (“shuffling”) of a part or parts of one or moreallelic forms of the gene of interest. Likewise, a naturally-occurringnucleic acid (e.g., a promoter) becomes isolated if it is introduced toa different locus of the genome. Nucleic acids which are “isolated,” asdefined herein, are also referred to as “heterologous” nucleic acids.See, e.g., Compounds and Methods for Site Directed Mutagenesis inEukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo HomologousSequence Targeting in Eukaryotic Cells, Zarling et al., WO 93/22443(PCT/US93/03868).

[0027] As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer, or chimeras thereof, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids).

[0028] By “nucleic acid library” is meant a collection of isolated DNAor RNA molecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism, tissue, or ofa cell type from that organism. Construction of exemplary nucleic acidlibraries, such as genomic and cDNA libraries, is taught in standardmolecular biology references such as Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol.152, AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al., MolecularCloning—A Laboratory Manual, 2nd ed., Vol.1-3 (1989); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., Eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1994).

[0029] As used herein “operably linked” includes reference to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame.

[0030] As used herein, the term “plant” includes reference to wholeplants, plant parts or organs (e.g., leaves, stems, roots, etc.), plantcells, seeds and progeny of same. Plant cell, as used herein, furtherincludes, without limitation, cells obtained from or found in: seeds,suspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. Plant cells can also be understood to include modifiedcells, such as protoplasts, obtained from the aforementioned tissues.The class of plants which can be used in the methods of the invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants. A particularly preferred plant is Zea mays.

[0031] As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or chimeras or analogsthereof that have the essential nature of a natural deoxy- orribo-nucleotide in that they hybridize, under stringent hybridizationconditions, to substantially the same nucleotide sequence as naturallyoccurring nucleotides and/or allow translation into the same aminoacid(s) as the naturally occurring nucleotide(s). A polynucleotide canbe full-length or a subsequence of a native or heterologous structuralor regulatory gene. Unless otherwise indicated, the term includesreference to the specified sequence as well as the complementarysequence thereof. Thus, DNAs or RNAs with backbones modified forstability or for other reasons are “polynucleotides” as that term isintended herein. Moreover, DNAs or RNAs comprising unusual bases, suchas inosine, or modified bases, such as tritylated bases, to name justtwo examples, are polynucleotides as the term is used herein. It will beappreciated that a great variety of modifications have been made to DNAand RNA that serve many useful purposes known to those of skill in theart. The term polynucleotide as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

[0032] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. Further, this invention contemplatesthe use of both the methionine-containing and the methionine-less aminoterminal variants of the protein of the invention.

[0033] As used herein “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, or seeds. Suchpromoters are referred to as “tissue preferred”. Promoters whichinitiate transcription only in certain tissue are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “repressible” promoter is apromoter which is under environmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue specific, tissuepreferred, cell type specific, and inducible promoters constitute theclass of “non-constitutive” promoters. A “constitutive” promoter is apromoter which is active under most environmental conditions.

[0034] As used herein “recombinant” includes reference to a cell orvector, that has been modified by the introduction of a heterologousnucleic acid or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed,under-expressed or not expressed at all as a result of humanintervention. The term “recombinant” as used herein does not encompassthe alteration of the cell or vector by naturally occurring events(e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout human intervention.

[0035] As used herein, a “recombinant expression cassette” is a nucleicacid construct, generated recombinantly or synthetically, with a seriesof specified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

[0036] The term “residue” or “amino acid residue” or “amino acid” areused interchangeably herein to refer to an amino acid that isincorporated into a protein, polypeptide, or peptide (collectively“protein”). The amino acid may be a naturally occurring amino acid and,unless otherwise limited, may encompass non-natural analogs of naturalamino acids that can function in a similar manner as naturally occurringamino acids.

[0037] The term “selectively hybridizes” includes reference tohybridization, under stringent hybridization conditions, of a nucleicacid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have about at least 80% sequenceidentity, preferably 90% sequence identity, and most preferably 100%sequence identity (i.e., complementary) with each other.

[0038] The term “stringent conditions” or “stringent hybridizationconditions” includes reference to conditions under which a probe willselectively hybridize to its target sequence, to a detectably greaterdegree than to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to the probe (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, optionally less than 500 nucleotides inlength.

[0039] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

[0040] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl, Anal. Biochem.,138:267-284 (1984): T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, %GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridizationand/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. Hybridizationand/or wash conditions can be applied for at least 10, 30, 60, 90, 120,or 240 minutes. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

[0041] As used herein, “transgenic plant” includes reference to a plantwhich comprises within its genome a heterologous polynucleotide.Generally, the heterologous polynucleotide is stably integrated withinthe genome such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant expression cassette.“Transgenic” is used herein to include any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of heterologous nucleic acid including those transgenicsinitially so altered as well as those created by sexual crosses orasexual propagation from the initial transgenic. The term “transgenic”as used herein does not encompass the alteration of the genome(chromosomal or extra-chromosomal) by conventional plant breedingmethods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

[0042] As used herein, “vector” includes reference to a nucleic acidused in introduction of a polynucleotide of the present invention into ahost cell. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

[0043] The following terms are used to describe the sequencerelationships between a polynucleotide/polypeptide of the presentinvention with a reference polynucleotide/polypeptide: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and (d)“percentage of sequence identity”.

[0044] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison with apolynucleotide/polypeptide of the present invention. A referencesequence may be a subset or the entirety of a specified sequence; forexample, as a segment of a full-length cDNA or gene sequence, or thecomplete cDNA or gene sequence.

[0045] (b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids residues in length, andoptionally can be 30, 40, 50, 100, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide/polypeptide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

[0046] Methods of alignment of sequences for comparison are well-knownin the art. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman, Adv.Appl. Math. 2: 482 (1981); by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444(1988); by computerized implementations of these algorithms, including,but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics,Mountain View, Calif.: GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis., USA; the CLUSTAL program is well describedby Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS5: 151-153 (1989); Corpet, et a., Nucleic Acids Research 16: 10881-90(1988); Huang, et al., ComputerApplications in the Biosciences 8: 155-65(1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331(1994).

[0047] The BLAST family of programs which can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995);Altschul et al., J. Mol. Biol., 215:403-410 (1990); and, Altschul etal., Nucleic Acids Res. 25:3389-3402 (1997).

[0048] Software for performing BLAST analyses is publicly available,e.g., through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

[0049] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5877 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

[0050] BLAST searches assume that proteins can be modeled as randomsequences. However, many real proteins comprise regions of nonrandomsequences which may be homopolymeric tracts, short-period repeats, orregions enriched in one or more amino acids. Such low-complexity regionsmay be aligned between unrelated proteins even though other regions ofthe protein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993))and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993))low-complexity filters can be employed alone or in combination.

[0051] Unless otherwise stated, nucleotide and proteinidentity/similarity values provided herein are calculated using GAP (GCGVersion 10) under default values.

[0052] GAP (Global Alignment Program) can also be used to compare apolynucleotide or polypeptide of the present invention with a referencesequence. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48: 443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

[0053] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0054] Multiple alignment of the sequences can be performed using theCLUSTAL method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Default parameters for pairwise alignments using the CLUSTAL method areKTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0055] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences includes referenceto the residues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

[0056] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

[0057] Utilities

[0058] The present invention provides, among other things, compositionsand methods for modulating (i.e., increasing or decreasing) the level ofpolynucleotides and polypeptides of the present invention in plants. Inparticular, the polynucleotides and polypeptides of the presentinvention can be expressed temporally or spatially, e.g., atdevelopmental stages, in tissues, and/or in quantities, which areuncharacteristic of non-recombinantly engineered plants.

[0059] The present invention also provides isolated nucleic acidscomprising polynucleotides of sufficient length and complementarity to apolynucleotide of the present invention to use as probes oramplification primers in the detection, quantitation, or isolation ofgene transcripts. For example, isolated nucleic acids of the presentinvention can be used as probes in detecting deficiencies in the levelof mRNA in screenings for desired transgenic plants, for detectingmutations in the gene (e.g., substitutions, deletions, or additions),for monitoring upregulation of expression or changes in enzyme activityin screening assays of compounds, for detection of any number of allelicvariants (polymorphisms), orthologs, or paralogs of the gene, or forsite directed mutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No.5,565,350). The isolated nucleic acids of the present invention can alsobe used for recombinant expression of their encoded polypeptides, or foruse as immunogens in the preparation and/or screening of antibodies. Theisolated nucleic acids of the present invention can also be employed foruse in sense or antisense suppression of one or more genes of thepresent invention in a host cell, tissue, or plant. Attachment ofchemical agents which bind, intercalate, cleave and/or crosslink to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation.

[0060] The present invention also provides isolated proteins comprisinga polypeptide of the present invention (e.g., preproenzyme, proenzyme,or enzymes). The present invention also provides proteins comprising atleast one epitope from a polypeptide of the present invention. Theproteins of the present invention can be employed in assays for enzymeagonists or antagonists of enzyme function, or for use as immunogens orantigens to obtain antibodies specifically immunoreactive with a proteinof the present invention. Such antibodies can be used in assays forexpression levels, for identifying and/or isolating nucleic acids of thepresent invention from expression libraries, for identification ofhomologous polypeptides from other species, or for purification ofpolypeptides of the present invention.

[0061] The isolated nucleic acids and polypeptides of the presentinvention can be used over a broad range of plant types, particularlymonocots such as the species of the family Gramineae including Hordeum,Secale, Oryza, Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z.mays), and dicots such as Glycine.

[0062] The isolated nucleic acid and proteins of the present inventioncan also be used in species from the genera: Cucurbita, Rosa, Vitis,Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis,Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browallia, Pisum, Phaseolus, Lolium, and Avena.

[0063] Nucleic Acids

[0064] The present invention provides, among other things, isolatednucleic acids of RNA, DNA, and analogs and/or chimeras thereof,comprising a polynucleotide of the present invention.

[0065] A polynucleotide of the present invention is inclusive of thosein Table 1 and:

[0066] (a) an isolated polynucleotide encoding a polypeptide of thepresent invention such as those referenced in Table 1, includingexemplary polynucleotides of the present invention;

[0067] (b) an isolated polynucleotide which is the product ofamplification from a plant nucleic acid library using primer pairs whichselectively hybridize under stringent conditions to loci within apolynucleotide of the present invention;

[0068] (c) an isolated polynucleotide which selectively hybridizes to apolynucleotide of (a) or (b);

[0069] (d) an isolated polynucleotide having a specified sequenceidentity with polynucleotides of (a), (b), or (c);

[0070] (e) an isolated polynucleotide encoding a protein having aspecified number of contiguous amino acids from a prototype polypeptide,wherein the protein is specifically recognized by antisera elicited bypresentation of the protein and wherein the protein does not detectablyimmunoreact to antisera which has been fully immunosorbed with theprotein;

[0071] (f) complementary sequences of polynucleotides of (a), (b), (c),(d), or (e); and

[0072] (g) an isolated polynucleotide comprising at least a specificnumber of contiguous nucleotides from a polynucleotide of (a), (b), (c),(d), (e), or (f);

[0073] (h) an isolated polynucleotide from a full-length enriched cDNAlibrary having the physico-chemical property of selectively hybridizingto a polynucleotide of (a), (b), (c), (d), (e), (f), or (g);

[0074] (i) an isolated polynucleotide made by the process of: 1)providing a full-length enriched nucleic acid library, 2) selectivelyhybridizing the polynucleotide to a polynucleotide of (a), (b), (c),(d), (e), (f), (g), or (h), thereby isolating the polynucleotide fromthe nucleic acid library.

[0075] A. Polynucleotides Encoding A Polypeptide of the PresentInvention

[0076] As indicated in (a), above, the present invention providesisolated nucleic acids comprising a polynucleotide of the presentinvention, wherein the polynucleotide encodes a polypeptide of thepresent invention. Every nucleic acid sequence herein that encodes apolypeptide also, by reference to the genetic code, describes everypossible silent variation of the nucleic acid. One of ordinary skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine; and UGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Thus, each silent variation of a nucleic acid whichencodes a polypeptide of the present invention is implicit in eachdescribed polypeptide sequence and is within the scope of the presentinvention. Accordingly, the present invention includes polynucleotidesof the present invention and polynucleotides encoding a polypeptide ofthe present invention.

[0077] B. Polynucleotides Amplified from a Plant Nucleic Acid Library

[0078] As indicated in (b), above, the present invention provides anisolated nucleic acid comprising a polynucleotide of the presentinvention, wherein the polynucleotides are amplified, under nucleic acidamplification conditions, from a plant nucleic acid library. Nucleicacid amplification conditions for each of the variety of amplificationmethods are well known to those of ordinary skill in the art. The plantnucleic acid library can be constructed from a monocot such as a cerealcrop. Exemplary cereals include maize, sorghum, alfalfa, canola, wheat,or rice. The plant nucleic acid library can also be constructed from adicot such as soybean. Zea mays lines B73, PHRE1, A632, BMS-P2#10, W23,and Mo17 are known and publicly available. Other publicly known andavailable maize lines can be obtained from the Maize GeneticsCooperation (Urbana, Ill.). Wheat lines are available from the WheatGenetics Resource Center (Manhattan, Kans.).

[0079] The nucleic acid library may be a cDNA library, a genomiclibrary, or a library generally constructed from nuclear transcripts atany stage of intron processing. cDNA libraries can be normalized toincrease the representation of relatively rare cDNAs. In optionalembodiments, the cDNA library is constructed using an enrichedfull-length cDNA synthesis method. Examples of such methods includeOligo-Capping (Maruyama, K. and Sugano, S. Gene 138: 171-174, 1994),Biotinylated CAP Trapper (Carninci, et al. Genomics 37: 327-336, 1996),and CAP Retention Procedure (Edery, E., Chu, L. L., et al. Molecular andCellular Biology 15: 3363-3371, 1995). Rapidly growing tissues orrapidly dividing cells are preferred for use as an mRNA source forconstruction of a cDNA library. Growth stages of maize are described in“How a Corn Plant Develops,” Special Report No. 48, Iowa StateUniversity of Science and Technology Cooperative Extension Service,Ames, Iowa, Reprinted February 1993.

[0080] A polynucleotide of this embodiment (or subsequences thereof) canbe obtained, for example, by using amplification primers which areselectively hybridized and primer extended, under nucleic acidamplification conditions, to at least two sites within a polynucleotideof the present invention, or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention, or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. Methods for obtaining 5′ and/or 3′ends of a vector insert are well known in the art. See, e.g., RACE(Rapid Amplification of Complementary Ends) as described in Frohman, M.A., in PCR Protocols: A Guide to Methods and Applications, M. A. Innis,D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc.,San Diego), pp. 28-38 (1990)); see also, U.S. Pat. No. 5,470,722, andCurrent Protocols in Molecular Biology, Unit 15.6, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995); Frohmanand Martin, Techniques 1:165 (1989).

[0081] Optionally, the primers are complementary to a subsequence of thetarget nucleic acid which they amplify but may have a sequence identityranging from about 85% to 99% relative to the polynucleotide sequencewhich they are designed to anneal to. As those skilled in the art willappreciate, the sites to which the primer pairs will selectivelyhybridize are chosen such that a single contiguous nucleic acid can beformed under the desired nucleic acid amplification conditions. Theprimer length in nucleotides is selected from the group of integersconsisting of from at least 15 to 50. Thus, the primers can be at least15, 18, 20, 25, 30, 40, or 50 nucleotides in length. Those of skill willrecognize that a lengthened primer sequence can be employed to increasespecificity of binding (i.e., annealing) to a target sequence. Anon-annealing sequence at the 5′ end of a primer (a “tail”) can beadded, for example, to introduce a cloning site at the terminal ends ofthe amplicon.

[0082] The amplification products can be translated using expressionsystems well known to those of skill in the art. The resultingtranslation products can be confirmed as polypeptides of the presentinvention by, for example, assaying for the appropriate catalyticactivity (e.g., specific activity and/or substrate specificity), orverifying the presence of one or more epitopes which are specific to apolypeptide of the present invention. Methods for protein synthesis fromPCR derived templates are known in the art and available commercially.See, e.g., Amersham Life Sciences, Inc, Catalog '97, p.354.

[0083] The polynucleotides of the present invention include thoseamplified using the following primer pairs:

[0084] SEQ ID NOS: 3 and 4, which yield an amplicon comprising asequence having substantial identity to SEQ ID NO: 1; and

[0085] SEQ ID NOS: 7 and 8, which yield an amplicon comprising asequence having substantial identity to SEQ ID NO: 5.

[0086] C. Polynucleotides Which Selectively Hybridize to aPolynucleotide of (A) or (B)

[0087] As indicated in (c), above, the present invention providesisolated nucleic acids comprising polynucleotides of the presentinvention, wherein the polynucleotides selectively hybridize, underselective hybridization conditions, to a polynucleotide of sections (A)or (B) as discussed above. Thus, the polynucleotides of this embodimentcan be used for isolating, detecting, and/or quantifying nucleic acidscomprising the polynucleotides of (A) or (B). For example,polynucleotides of the present invention can be used to identify,isolate, or amplify partial or full-length clones in a depositedlibrary. In some embodiments, the polynucleotides are genomic or cDNAsequences isolated or otherwise complementary to a cDNA from a dicot ormonocot nucleic acid library. Exemplary species of monocots and dicotsinclude, but are not limited to: maize, canola, soybean, cotton, wheat,sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and rice.The cDNA library comprises at least 50% to 95% full-length sequences(for example, at least 50%, 60%, 70%, 80%, 90%, or 95% full-lengthsequences). The cDNA libraries can be normalized to increase therepresentation of rare sequences. See, e.g., U.S. Pat. No. 5,482,845.Low stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% to 80% sequence identity and can be employed toidentify orthologous or paralogous sequences.

[0088] D. Polynucleotides Having a Specific Sequence Identity with thePolynucleotides of (A), (B) or (C)

[0089] As indicated in (d), above, the present invention providesisolated nucleic acids comprising polynucleotides of the presentinvention, wherein the polynucleotides have a specified identity at thenucleotide level to a polynucleotide as disclosed above in sections (A),(B), or (C), above. Identity can be calculated using, for example, theBLAST, CLUSTALW, or GAP algorithms under default conditions. Thepercentage of identity to a reference sequence is at least 50% and,rounded upwards to the nearest integer, can be expressed as an integerselected from the group of integers consisting of from 50 to 99. Thus,for example, the percentage of identity to a reference sequence can beat least 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0090] Optionally, the polynucleotides of this embodiment will encode apolypeptide that will share an epitope with a polypeptide encoded by thepolynucleotides of sections (A), (B), or (C). Thus, thesepolynucleotides encode a first polypeptide which elicits production ofantisera comprising antibodies which are specifically reactive to asecond polypeptide encoded by a polynucleotide of (A), (B), or (C).However, the first polypeptide does not bind to antisera raised againstitself when the antisera has been fully immunosorbed with the firstpolypeptide. Hence, the polynucleotides of this embodiment can be usedto generate antibodies for use in, for example, the screening ofexpression libraries for nucleic acids comprising polynucleotides of(A), (B), or (C), or for purification of, or in immunoassays for,polypeptides encoded by the polynucleotides of (A), (B), or (C). Thepolynucleotides of this embodiment comprise nucleic acid sequences whichcan be employed for selective hybridization to a polynucleotide encodinga polypeptide of the present invention.

[0091] Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure. Antibody screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 15amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT patent publication Nos.91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent publication Nos. 92/05258,92/14843, and 97/20078. See also, U.S. Pat. Nos. 5,658,754; and5,643,768. Peptide display libraries, vectors, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.).

[0092] E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and Cross-Reactive to the Prototype Polypeptide

[0093] As indicated in (e), above, the present invention providesisolated nucleic acids comprising polynucleotides of the presentinvention, wherein the polynucleotides encode a protein having asubsequence of contiguous amino acids from a prototype polypeptide ofthe present invention such as are provided in (a), above. The length ofcontiguous amino acids from the prototype polypeptide is selected fromthe group of integers consisting of from at least 10 to the number ofamino acids within the prototype sequence. Thus, for example, thepolynucleotide can encode a polypeptide having a subsequence having atleast 10, 15, 20, 25, 30, 35, 40, 45, or 50, contiguous amino acids fromthe prototype polypeptide. Further, the number of such subsequencesencoded by a polynucleotide of the instant embodiment can be any integerselected from the group consisting of from 1 to 20, such as 2, 3, 4, or5. The subsequences can be separated by any integer of nucleotides from1 to the number of nucleotides in the sequence such as at least 5, 10,15, 25, 50, 100, or 200 nucleotides.

[0094] The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such asbut not limited to, a polypeptide encoded by the polynucleotide of (a)or (b), above. Generally, however, a protein encoded by a polynucleotideof this embodiment does not bind to antisera raised against theprototype polypeptide when the antisera has been fully immunosorbed withthe prototype polypeptide. Methods of making and assaying for antibodybinding specificity/affinity are well known in the art. Exemplaryimmunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

[0095] In a preferred assay method, fully immunosorbed and pooledantisera which is elicited to the prototype polypeptide can be used in acompetitive binding assay to test the protein. The concentration of theprototype polypeptide required to inhibit 50% of the binding of theantisera to the prototype polypeptide is determined. If the amount ofthe protein required to inhibit binding is less than twice the amount ofthe prototype protein, then the protein is said to specifically bind tothe antisera elicited to the immunogen. Accordingly, the proteins of thepresent invention embrace allelic variants, conservatively modifiedvariants, and minor recombinant modifications to a prototypepolypeptide.

[0096] A polynucleotide of the present invention optionally encodes aprotein having a molecular weight as the non-glycosylated protein within20% of the molecular weight of the full-length non-glycosylatedpolypeptides of the present invention. Molecular weight can be readilydetermined by SDS-PAGE under reducing conditions. Optionally, themolecular weight is within 15% of a full length polypeptide of thepresent invention, more preferably within 10% or 5%, and most preferablywithin 3%, 2%, or 1% of a full length polypeptide of the presentinvention.

[0097] Optionally, the polynucleotides of this embodiment will encode aprotein having a specific enzymatic activity at least 50%, 60%, 80%, or90% of a cellular extract comprising the native, endogenous full-lengthpolypeptide of the present invention. Further, the proteins encoded bypolynucleotides of this embodiment will optionally have a substantiallysimilar affinity constant (K_(m)) and/or catalytic activity (i.e., themicroscopic rate constant, k_(cat)) as the native endogenous,full-length protein. Those of skill in the art will recognize thatk_(cat)/K_(m) value determines the specificity for competing substratesand is often referred to as the specificity constant. Proteins of thisembodiment can have a k_(cat)/K_(m) value at least 10% of a full-lengthpolypeptide of the present invention as determined using the endogenoussubstrate of that polypeptide. Optionally, the k_(cat)/K_(m) value willbe at least 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%,80%, 90%, or 95% the k_(cat)/K_(m) value of the full-length polypeptideof the present invention. Determination of k_(cat), K_(m), andk_(cat)/K_(m) can be determined by any number of means well known tothose of skill in the art. For example, the initial rates (i.e., thefirst 5% or less of the reaction) can be determined using rapid mixingand sampling techniques (e.g., continuous-flow, stopped-flow, or rapidquenching techniques), flash photolysis, or relaxation methods (e.g.,temperature jumps) in conjunction with such exemplary methods ofmeasuring as spectrophotometry, spectrofluorimetry, nuclear magneticresonance, or radioactive procedures. Kinetic values are convenientlyobtained using a Lineweaver-Burk or Eadie-Hofstee plot.

[0098] F. Polynucleotides Complementary to the Polynucleotides of(A)-(E)

[0099] As indicated in (f), above, the present invention providesisolated nucleic acids comprising polynucleotides complementary to thepolynucleotides of paragraphs A-E, above. As those of skill in the artwill recognize, complementary sequences base-pair throughout theentirety of their length with the polynucleotides of sections (A)-(E)(i.e., have 100% sequence identity over their entire length).Complementary bases associate through hydrogen bonding in doublestranded nucleic acids. For example, the following base pairs arecomplementary: guanine and cytosine; adenine and thymine; and adenineand uracil.

[0100] G. Polynucleotides Which are Subsequences of the Polynucleotidesof (A)-(F)

[0101] As indicated in (g), above, the present invention providesisolated nucleic acids comprising polynucleotides which comprise atleast 15 contiguous bases from the polynucleotides of sections (A)through (F) as discussed above. The length of the polynucleotide isgiven as an integer selected from the group consisting of from at least15 to the length of the nucleic acid sequence from which thepolynucleotide is a subsequence of. Thus, for example, polynucleotidesof the present invention are inclusive of polynucleotides comprising atleast 15, 20, 25, 30, 40, 50, 60, 75, or 100 contiguous nucleotides inlength from the polynucleotides of (A)-(F). Optionally, the number ofsuch subsequences encoded by a polynucleotide of the instant embodimentcan be any integer selected from the group consisting of from 1 to 20,such as 2, 3, 4, or 5. The subsequences can be separated by any integerof nucleotides from 1 to the number of nucleotides in the sequence suchas at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

[0102] Subsequences can be made by in vitro synthetic, in vitrobiosynthetic, or in vivo recombinant methods. In optional embodiments,subsequences can be made by nucleic acid amplification. For example,nucleic acid primers will be constructed to selectively hybridize to asequence (or its complement) within, or co-extensive with, the codingregion.

[0103] The subsequences of the present invention can comprise structuralcharacteristics of the sequence from which it is derived. Alternatively,the subsequences can lack certain structural characteristics of thelarger sequence from which it is derived such as a poly (A) tail.Optionally, a subsequence from a polynucleotide encoding a polypeptidehaving at least one epitope in common with a prototype polypeptidesequence as provided in (a), above, may encode an epitope in common withthe prototype sequence. Alternatively, the subsequence may not encode anepitope in common with the prototype sequence but can be used to isolatethe larger sequence by, for example, nucleic acid hybridization with thesequence from which it's derived. Subsequences can be used to modulateor detect gene expression by introducing into the subsequences compoundswhich bind, intercalate, cleave and/or crosslink to nucleic acids.Exemplary compounds include acridine, psoralen, phenanthroline,naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.

[0104] H. Polynucleotides From a Full-length Enriched cDNA LibraryHaving the Physico-Chemical Property of Selectively Hybridizing to aPolynucleotide of (A)-(G)

[0105] As indicated in (h), above, the present invention provides anisolated polynucleotide from a full-length enriched cDNA library havingthe physico-chemical property of selectively hybridizing to apolynucleotide of paragraphs (A), (B), (C), (D), (E), (F), or (G) asdiscussed above. Methods of constructing full-length enriched cDNAlibraries are known in the art and discussed briefly below. The cDNAlibrary comprises at least 50% to 95% full-length sequences (forexample, at least 50%, 60%, 70%, 80%, 90%, or 95% full-lengthsequences). The cDNA library can be constructed from a variety oftissues from a monocot or dicot at a variety of developmental stages.Exemplary species include maize, wheat, rice, canola, soybean, cotton,sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and rice.Methods of selectively hybridizing, under selective hybridizationconditions, a polynucleotide from a full-length enriched library to apolynucleotide of the present invention are known to those of ordinaryskill in the art. Any number of stringency conditions can be employed toallow for selective hybridization. In optional embodiments, thestringency allows for selective hybridization of sequences having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity over thelength of the hybridized region. Full-length enriched cDNA libraries canbe normalized to increase the representation of rare sequences.

[0106] I. Polynucleotide Products Made by a cDNA Isolation Process

[0107] As indicated in (I), above, the present invention provides anisolated polynucleotide made by the process of: 1) providing afull-length enriched nucleic acid library, 2) selectively hybridizingthe polynucleotide to a polynucleotide of paragraphs (A), (B), (C), (D),(E), (F), (G, or (H) as discussed above, and thereby isolating thepolynucleotide from the nucleic acid library. Full-length enrichednucleic acid libraries are constructed as discussed in paragraph (G) andbelow. Selective hybridization conditions are as discussed in paragraph(G). Nucleic acid purification procedures are well known in the art.Purification can be conveniently accomplished using solid-phase methods;such methods are well known to those of skill in the art and kits areavailable from commercial suppliers such as Advanced Biotechnologies(Surrey, UK). For example, a polynucleotide of paragraphs (A)-(H) can beimmobilized to a solid support such as a membrane, bead, or particle.See, e.g., U.S. Pat. No. 5,667,976. The polynucleotide product of thepresent process is selectively hybridized to an immobilizedpolynucleotide and the solid support is subsequently isolated fromnon-hybridized polynucleotides by methods including, but not limited to,centrifugation, magnetic separation, filtration, electrophoresis, andthe like.

[0108] Construction of Nucleic Acids

[0109] The isolated nucleic acids of the present invention can be madeusing (a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot such as maize, rice, or wheat, or a dicot such assoybean.

[0110] The nucleic acids may conveniently comprise sequences in additionto a polynucleotide of the present invention. For example, amulti-cloning site comprising one or more endonuclease restriction sitesmay be inserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. A polynucleotideof the present invention can be attached to a vector, adapter, or linkerfor cloning and/or expression of a polynucleotide of the presentinvention. Additional sequences may be added to such cloning and/orexpression sequences to optimize their function in cloning and/orexpression, to aid in isolation of the polynucleotide, or to improve theintroduction of the polynucleotide into a cell. Typically, the length ofa nucleic acid of the present invention less the length of itspolynucleotide of the present invention is less than 20 kilobase pairs,often less than 15 kb, and frequently less than 10 kb. Use of cloningvectors, expression vectors, adapters, and linkers is well known andextensively described in the art. For a description of various nucleicacids see, for example, Stratagene Cloning Systems, Catalogs 1999 (LaJolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '99 (ArlingtonHeights, Ill.).

[0111] A. Recombinant Methods for Constructing Nucleic Acids

[0112] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. Isolation of RNA, andconstruction of cDNA and genomic libraries is well known to those ofordinary skill in the art. See, e.g., Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and,Current Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

[0113] A1. Full-length Enriched cDNA Libraries

[0114] A number of cDNA synthesis protocols have been described whichprovide enriched full-length cDNA libraries. Enriched full-length cDNAlibraries are constructed to comprise at least 600%, and more preferablyat least 70%, 80%, 90% or 95% full-length inserts amongst clonescontaining inserts. The length of insert in such libraries can be atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors toaccommodate inserts of these sizes are known in the art and availablecommercially. See, e.g., Stratagene's lambda ZAP Express (cDNA cloningvector with 0 to 12 kb cloning capacity). An exemplary method ofconstructing a greater than 95% pure full-length cDNA library isdescribed by Carninci et al., Genomics, 37:327-336 (1996). Other methodsfor producing full-length libraries are known in the art. See, e.g.,Edery et al., Mol. Cell Biol., 15(6):3363-3371 (1995); and, PCTApplication WO 96/34981.

[0115] A2 Normalized or Subtracted cDNA Libraries

[0116] A non-normalized cDNA library represents the mRNA population ofthe tissue it was made from. Since unique clones are out-numbered byclones derived from highly expressed genes their isolation can belaborious. Normalization of a cDNA library is the process of creating alibrary in which each clone is more equally represented. Construction ofnormalized libraries is described in Ko, Nucl. Acids. Res.,18(19):5705-5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A.,88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685, 5,482,845, and 5,637,685.In an exemplary method described by Soares et al., normalizationresulted in reduction of the abundance of clones from a range of fourorders of magnitude of a narrow range of only 1 order of magnitude.Proc. Natl. Acad. Sci. USA, 91:9228-9232 (1994).

[0117] Subtracted cDNA libraries are another means to increase theproportion of less abundant cDNA species. In this procedure, cDNAprepared from one pool of mRNA is depleted of sequences present in asecond pool of mRNA by hybridization. The cDNA:mRNA hybrids are removedand the remaining un-hybridized cDNA pool is enriched for sequencesunique to that pool. See, Foote et al. in, Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho andZarbl, Technique, 3(2):58-63 (1991); Sive and St. John, Nucl. AcidsRes., 16(22):10937 (1988); Current Protocols in Molecular Biology,Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, NewYork (1995); and, Swaroop et al., Nucl. Acids Res., 19)8):1954 (1991).cDNA subtraction kits are commercially available. See, e.g., PCR-Select(Clontech, Palo Alto, Calif.).

[0118] To construct genomic libraries, large segments of genomic DNA aregenerated by fragmentation, e.g. using restriction endonucleases, andare ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends, andsequencing methods to verify the sequence of nucleic acids are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning, and screening methodologies arefound in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods inEnzymology, Vol.152: Guide to Molecular Cloning Techniques, Berger andKimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocolsin Molecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

[0119] The cDNA or genomic library can be screened using a probe basedupon the sequence of a polynucleotide of the present invention such asthose disclosed herein. Probes may be used to hybridize with genomic DNAor cDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent.

[0120] The nucleic acids of interest can also be amplified from nucleicacid samples using amplification techniques. For instance, polymerasechain reaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

[0121] PCR-based screening methods have been described. Wilfinger et al.describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.Bio Techniques, 22(3): 481-486 (1997). Such methods are particularlyeffective in combination with a full-length cDNA constructionmethodology, above.

[0122] B. Synthetic Methods for Constructing Nucleic Acids

[0123] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68: 90-99(1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage et al.,Tetra. Lett. 22: 1859-1862 (1981); the solid phase phosphoramiditetriester method described by Beaucage and Caruthers, Tetra. Letts.22(20): 1859-1862 (1981), e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984); and, the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis generally produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill willrecognize that while chemical synthesis of DNA is best employed forsequences of about 100 bases or less, longer sequences may be obtainedby the ligation of shorter sequences.

[0124] Recombinant Expression Cassettes

[0125] The present invention further provides recombinant expressioncassettes comprising a nucleic acid of the present invention. A nucleicacid sequence coding for the desired polypeptide of the presentinvention, for example a cDNA or a genomic sequence encoding a fulllength polypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

[0126] For example, plant expression vectors may include (1) a clonedplant gene under the transcriptional control of 5′ and 3′ regulatorysequences and (2) a dominant selectable marker. Such plant expressionvectors may also contain, if desired, a promoter regulatory region(e.g., one conferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0127] A plant promoter fragment can be employed which will directexpression of a polynucleotide of the present invention in all tissuesof a regenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter,and the GRP1-8 promoter.

[0128] Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight.

[0129] Examples of promoters under developmental control includepromoters that initiate transcription only, or preferentially, incertain tissues, such as leaves, roots, fruit, seeds, or flowers.Exemplary promoters include the anther-specific promoter 5126 (U.S. Pat.Nos. 5,689,049 and 5,689,051), glb-1 promoter, and gamma-zein promoter.Also see, for example, U.S. patent applications Nos. 60/155,859, and60/163,114. The operation of a promoter may also vary depending on itslocation in the genome. Thus, an inducible promoter may become fully orpartially constitutive in certain locations.

[0130] Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inrecombinant expression cassettes to drive expression of antisensenucleic acids to reduce, increase, or alter concentration and/orcomposition of the proteins of the present invention in a desiredtissue. Thus, in some embodiments, the nucleic acid construct willcomprise a promoter, functional in a plant cell, operably linked to apolynucleotide of the present invention. Promoters useful in theseembodiments include the endogenous promoters driving expression of apolypeptide of the present invention.

[0131] In some embodiments, isolated nucleic acids which serve aspromoter or enhancer elements can be introduced in the appropriateposition (generally upstream) of a non-heterologous form of apolynucleotide of the present invention so as to up or down regulateexpression of a polynucleotide of the present invention. For example,endogenous promoters can be altered in vivo by mutation, deletion,and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling etal., PCT/US93/03868), or isolated promoters can be introduced into aplant cell in the proper orientation and distance from a cognate gene ofa polynucleotide of the present invention so as to control theexpression of the gene. Gene expression can be modulated underconditions suitable for plant growth so as to alter the totalconcentration and/or alter the composition of the polypeptides of thepresent invention in plant cell. Thus, the present invention providescompositions, and methods for making, heterologous promoters and/orenhancers operably linked to a native, endogenous (i.e.,non-heterologous) form of a polynucleotide of the present invention.

[0132] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0133] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold. Buchman and Berg,Mol. Cell Biol. 8: 4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression istypically greatest when placed near the 5′ end of the transcriptionunit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1intron are known in the art. See generally, The Maize Handbook, Chapter116, Freeling and Walbot, Eds., Springer, New York (1994). The vectorcomprising the sequences from a polynucleotide of the present inventionwill typically comprise a marker gene which confers a selectablephenotype on plant cells. Typical vectors useful for expression of genesin higher plants are well known in the art and include vectors derivedfrom the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciensdescribed by Rogers et al., Meth. in Enzymol., 153:253-277 (1987).

[0134] A polynucleotide of the present invention can be expressed ineither sense or anti-sense orientation as desired. It will beappreciated that control of gene expression in either sense oranti-sense orientation can have a direct impact on the observable plantcharacteristics. Antisense technology can be conveniently used toinhibit gene expression in plants. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed. Theconstruct is then transformed into plants and the antisense strand ofRNA is produced. In plant cells, it has been shown that antisense RNAinhibits gene expression by preventing the accumulation of mRNA whichencodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Nat'l.Acad. Sci. (USA) 85: 8805-8809 (1988); and Hiatt et al., U.S. Pat.No.4,801,340.

[0135] Another method of suppression is sense suppression (i.e.,co-supression). Introduction of nucleic acid configured in the senseorientation has been shown to be an effective means by which to blockthe transcription of target genes. For an example of the use of thismethod to modulate expression of endogenous genes see, Napoli et al.,The Plant Cell 2: 279-289 (1990) and U.S. Pat. No. 5,034,323.

[0136] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al., Nature334: 585-591 (1988).

[0137] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation b a modified nucleotide which was capable of activatingcleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B., et al., JAm Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to atarget nucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203.Use of crosslinking in triple-helix forming probes was also disclosed byHome, et al., J Am Chem Soc (1990) 112:2435-2437. Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to single-strandedoligonucleotides has also been described by Webb and Matteucci, J AmChem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674;Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds tobind, detect, label, and/or cleave nucleic acids are known in the art.See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908;5,256,648; and, 5,681941.

[0138] Proteins

[0139] The isolated proteins of the present invention comprise apolypeptide having at least 10 amino acids from a polypeptide of thepresent invention (or conservative variants thereof such as thoseencoded by any one of the polynucleotides of the present invention asdiscussed more fully above (e.g., Table 1). The proteins of the presentinvention or variants thereof can comprise any number of contiguousamino acid residues from a polypeptide of the present invention, whereinthat number is selected from the group of integers consisting of from 10to the number of residues in a full-length polypeptide of the presentinvention. Optionally, this subsequence of contiguous amino acids is atleast 15, 20, 25, 30, 35, or 40 amino acids in length, often at least50, 60, 70, 80, or 90 amino acids in length. Further, the number of suchsubsequences can be any integer selected from the group consisting offrom 1 to 20, such as 2, 3, 4, or 5.

[0140] The present invention further provides a protein comprising apolypeptide having a specified sequence identity/similarity with apolypeptide of the present invention. The percentage of sequenceidentity/similarity is an integer selected from the group consisting offrom 50 to 99. Exemplary sequence identity/similarity values include55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Sequence identity canbe determined using, for example, the GAP, CLUSTALW, or BLASTalgorithms.

[0141] As those of skill will appreciate, the present inventionincludes, but is not limited to, catalytically active polypeptides ofthe present invention (i.e., enzymes). Catalytically active polypeptideshave a specific activity of at least 20%, 30%, or 40%, and preferably atleast 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%that of the native (non-synthetic), endogenous polypeptide. Further, thesubstrate specificity (k_(cat)/K_(m)) is optionally substantiallysimilar to the native (non-synthetic), endogenous polypeptide.Typically, the K_(m) will be at least 30%, 40%, or 50%, that of thenative (non-synthetic), endogenous polypeptide; and more preferably atleast 60%, 70%, 80%, or 90%. Methods of assaying and quantifyingmeasures of enzymatic activity and substrate specificity(k_(cat)/K_(m)), are well known to those of skill in the art.

[0142] Generally, the proteins of the present invention will, whenpresented as an immunogen, elicit production of an antibody specificallyreactive to a polypeptide of the present invention. Further, theproteins of the present invention will not bind to antisera raisedagainst a polypeptide of the present invention which has been fullyimmunosorbed with the same polypeptide. Immunoassays for determiningbinding are well known to those of skill in the art. A preferredimmunoassay is a competitive immunoassay. Thus, the proteins of thepresent invention can be employed as immunogens for constructingantibodies immunoreactive to a protein of the present invention for suchexemplary utilities as immunoassays or protein purification techniques.

[0143] Expression of Proteins in Host Cells

[0144] Using the nucleic acids of the present invention, one may expressa protein of the present invention in a recombinantly engineered cellsuch as bacteria, yeast, insect, mammalian, or preferably plant cells.The cells produce the protein in a non-natural condition (e.g., inquantity, composition, location, and/or time), because they have beengenetically altered through human intervention to do so.

[0145] It is expected that those of skill in the art are knowledgeablein the numerous expression systems available for expression of a nucleicacid encoding a protein of the present invention. No attempt to describein detail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

[0146] In brief summary, the expression of isolated nucleic acidsencoding a protein of the present invention will typically be achievedby operably linking, for example, the DNA or cDNA to a promoter (whichis either constitutive or regulatable), followed by incorporation intoan expression vector. The vectors can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of theDNA encoding a protein of the present invention. To obtain high levelexpression of a cloned gene, it is desirable to construct expressionvectors which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation terminator. One of skill would recognizethat modifications can be made to a protein of the present inventionwithout diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, amethionine added at the amino terminus to provide an initiation site, oradditional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located purification sequences. Restriction sites ortermination codons can also be introduced.

[0147] Synthesis of Proteins

[0148] The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield, et al., J. Am. Chem. Soc. 85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nded., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater lengthmay be synthesized by condensation of the amino and carboxy termini ofshorter fragments. Methods of forming peptide bonds by activation of acarboxy terminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

[0149] Purification of Proteins

[0150] The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

[0151] The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. The protein may then be isolatedfrom cells expressing the protein and further purified by standardprotein chemistry techniques as described herein. Detection of theexpressed protein is achieved by methods known in the art and include,for example, radioimmunoassays, Western blotting techniques orimmunoprecipitation.

[0152] Introduction of Nucleic Acids Into Host Cells

[0153] The method of introducing a nucleic acid of the present inventioninto a host cell is not critical to the instant invention.Transformation or transfection methods are conveniently used.Accordingly, a wide variety of methods have been developed to insert aDNA sequence into the genome of a host cell to obtain the transcriptionand/or translation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for effective introduction ofa nucleic acid may be employed.

[0154] A. Plant Transformation

[0155] A nucleic acid comprising a polynucleotide of the presentinvention is optionally introduced into a plant. Generally, thepolynucleotide will first be incorporated into a recombinant expressioncassette or vector. Isolated nucleic acid acids of the present inventioncan be introduced into plants according to techniques known in the art.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical, scientific, and patentliterature. See, for example, Weising et al., Ann. Rev. Genet. 22:421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, polyethylene glycol (PEG) poration, particlebombardment, silicon fiber delivery, or microinjection of plant cellprotoplasts or embryogenic callus. See, e.g., Tomes, et al., Direct DNATransfer into Intact Plant Cells Via Microprojectile Bombardment.pp.197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods.eds. O. L. Gamborg and G. C. Phillips. Springer-Verlag Berlin HeidelbergNew York, 1995; see, U.S. Pat. No. 5,990,387. The introduction of DNAconstructs using PEG precipitation is described in Paszkowski et al.,Embo J. 3: 2717-2722 (1984). Electroporation techniques are described inFrom et al., Proc. Natl. Acad. Sci. (USA) 82: 5824 (1985). Ballistictransformation techniques are described in Klein et al., Nature 327:70-73 (1987).

[0156]Agrobacterium tumefaciens-mediated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233: 496-498 (1984); Fraley et al., Proc. Natl. Acad. Sci.(USA) 80: 4803 (1983); and, Plant Molecular Biology: A LaboratoryManual, Chapter 8, Clark, Ed., Springer-Verlag, Berlin (1997). The DNAconstructs may be combined with suitable T-DNA flanking regions andintroduced into a conventional Agrobacterium tumefaciens host vector.The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria. See, U.S. Pat. No.5,591,616. Although Agrobacterium is useful primarily in dicots, certainmonocots can be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

[0157] Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, PWJ Rigby, Ed.,London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J,.In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985),Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman et al., Plant Cell Physiol. 25: 1353 (1984)),(3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci.,(USA) 87: 1228 (1990).

[0158] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codinggenes can be obtained by injection of the DNA into reproductive organsof a plant as described by Pena et al., Nature, 325.:274 (1987). DNA canalso be injected directly into the cells of immature embryos and therehydration of desiccated embryos as described by Neuhaus et al., Theor.Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plantviruses that can be employed as vectors are known in the art and includecauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, andtobacco mosaic virus.

[0159] B. Transfection of Prokaryotes, Lower Eukaryotes, and AnimalCells

[0160] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transfection by various means. Thereare several well-known methods of introducing DNA into animal cells.These include: calcium phosphate precipitation, fusion of the recipientcells with bacterial protoplasts containing the DNA, treatment of therecipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

[0161] Transgenic Plant Regeneration

[0162] Plant cells which directly result or are derived from the nucleicacid introduction techniques can be cultured to regenerate a whole plantwhich possesses the introduced genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium. Plants cells can be regenerated, e.g., from single cells,callus tissue or leaf discs according to standard plant tissue culturetechniques. It is well known in the art that various cells, tissues, andorgans from almost any plant can be successfully cultured to regeneratean entire plant. Plant regeneration from cultured protoplasts isdescribed in Evans et al., Protoplasts Isolation and Culture, Handbookof Plant Cell Culture, Macmillan Publishing Company, New York, pp.124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts,CRC Press, Boca Raton, pp. 21-73 (1985).

[0163] The regeneration of plants from either single plant protoplastsor various explants is well known in the art. See, for example, Methodsfor Plant Molecular Biology, A. Weissbach and H. Weissbach, eds.,Academic Press, Inc., San Diego, Calif. (1988). This regeneration andgrowth process includes the steps of selection of transformant cells andshoots, rooting the transformant shoots and growth of the plantlets insoil. For maize cell culture and regeneration see generally, The MaizeHandbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn andCorn Improvement, 3^(rd) edition, Sprague and Dudley Eds., AmericanSociety of Agronomy, Madison, Wis. (1988). For transformation andregeneration of maize see, Gordon-Kamm et al., The Plant Cell, 2:603-618(1990).

[0164] The regeneration of plants containing the polynucleotide of thepresent invention and introduced by Agrobacterium from leaf explants canbe achieved as described by Horsch et al., Science, 227:1229-1231(1985). In this procedure, transformants are grown in the presence of aselection agent and in a medium that induces the regeneration of shootsin the plant species being transformed as described by Fraley et al.,Proc. Natl. Acad. Sci. (U.S.A.), 80:4803 (1983). This proceduretypically produces shoots within two to four weeks and thesetransformant shoots are then transferred to an appropriate root-inducingmedium containing the selective agent and an antibiotic to preventbacterial growth. Transgenic plants of the present invention may befertile or sterile.

[0165] One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype. Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences. Transgenic plants expressing a polynucleotide ofthe present invention can be screened for transmission of the nucleicacid of the present invention by, for example, standard immunoblot andDNA detection techniques. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then analyzed for protein expression by Western immunoblot analysisusing the specifically reactive antibodies of the present invention. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

[0166] A preferred embodiment is a transgenic plant that is homozygousfor the added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

[0167] Modulating Polypeptide Levels and/or Composition

[0168] The present invention further provides a method for modulating(i.e., increasing or decreasing) the concentration or ratio of thepolypeptides of the present invention in a plant or part thereof.Modulation can be effected by increasing or decreasing the concentrationand/or the ratio of the polypeptides of the present invention in aplant. The method comprises introducing into a plant cell a recombinantexpression cassette comprising a polynucleotide of the present inventionas described above to obtain a transgenic plant cell, culturing thetransgenic plant cell under transgenic plant cell growing conditions,and inducing or repressing expression of a polynucleotide of the presentinvention in the transgenic plant for a time sufficient to modulateconcentration and/or the ratios of the polypeptides in the transgenicplant or plant part.

[0169] In some embodiments, the concentration and/or ratios ofpolypeptides of the present invention in a plant may be modulated byaltering, in vivo or in vitro, the promoter of a gene to up- ordown-regulate gene expression. In some embodiments, the coding regionsof native genes of the present invention can be altered viasubstitution, addition, insertion, or deletion to decrease activity ofthe encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarlinget al., PCT/US93/03868. And in some embodiments, an isolated nucleicacid (e.g., a vector) comprising a promoter sequence is transfected intoa plant cell. Subsequently, a plant cell comprising the promoteroperably linked to a polynucleotide of the present invention is selectedfor by means known to those of skill in the art such as, but not limitedto, Southern blot, DNA sequencing, or PCR analysis using primersspecific to the promoter and to the gene and detecting ampliconsproduced therefrom. A plant or plant part altered or modified by theforegoing embodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or ratios of polypeptidesof the present invention in the plant. Plant forming conditions are wellknown in the art and discussed briefly, supra.

[0170] In general, concentration or the ratios of the polypeptides isincreased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% relative to a native control plant, plant part, or celllacking the aforementioned recombinant expression cassette. Modulationin the present invention may occur during and/or subsequent to growth ofthe plant to the desired stage of development. Modulating nucleic acidexpression temporally and/or in particular tissues can be controlled byemploying the appropriate promoter operably linked to a polynucleotideof the present invention in, for example, sense or antisense orientationas discussed in greater detail, supra. Induction of expression of apolynucleotide of the present invention can also be controlled byexogenous administration of an effective amount of inducing compound.Inducible promoters and inducing compounds which activate expressionfrom these promoters are well known in the art. In preferredembodiments, the polypeptides of the present invention are modulated inmonocots, particularly maize.

[0171] UTRs and Codon Preference

[0172] In general, translational efficiency has been found to beregulated by specific sequence elements in the 5′ non-coding oruntranslated region (5′ UTR) of the RNA. Positive sequence motifsinclude translational initiation consensus sequences (Kozak, NucleicAcids Res. 15:8125 (1987)) and the 7-methylguanosine cap structure(Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elementsinclude stable intramolecular 5′ UTR stem-loop structures (Muesing etal., Cell 48:691 (1987)) and AUG sequences or short open reading framespreceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al.,Mol. and Cell. Biol. 8:284 (1988)). Accordingly, the present inventionprovides 5′ and/or 3′ untranslated regions for modulation of translationof heterologous coding sequences.

[0173] Further, the polypeptide-encoding segments of the polynucleotidesof the present invention can be modified to alter codon usage. Alteredcodon usage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host such as tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see Devereaux etal., Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50, or 100.

[0174] Sequence Shuffling

[0175] The present invention provides methods for sequence shufflingusing polynucleotides of the present invention, and compositionsresulting therefrom. Sequence shuffling is described in PCT publicationNo. WO 97/20078. See also, Zhang, J.- H., et al. Proc. Natl. Acad. Sci.USA 94:4504-4509 (1997). Generally, sequence shuffling provides a meansfor generating libraries of polynucleotides having a desiredcharacteristic which can be selected or screened for. Libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides which comprise sequence regions which havesubstantial sequence identity and can be homologously recombined invitro or in vivo. The population of sequence-recombined polynucleotidescomprises a subpopulation of polynucleotides which possess desired oradvantageous characteristics and which can be selected by a suitableselection or screening method. The characteristics can be any propertyor attribute capable of being selected for or detected in a screeningsystem, and may include properties of: an encoded protein, atranscriptional element, a sequence controlling transcription, RNAprocessing, RNA stability, chromatin conformation, translation, or otherexpression property of a gene or transgene, a replicative element, aprotein-binding element, or the like, such as any feature which confersa selectable or detectable property. In some embodiments, the selectedcharacteristic will be a decreased K_(m) and/or increased K_(cat) overthe wild-type protein as provided herein. In other embodiments, aprotein or polynucleotide generated from sequence shuffling will have aligand binding affinity greater than the non-shuffled wild-typepolynucleotide. The increase in such properties can be at least 110%,120%, 130%, 140% or at least 150% of the wild-type value.

[0176] Generic and Consensus Sequences

[0177] Polynucleotides and polypeptides of the present invention furtherinclude those having: (a) a generic sequence of at least two homologouspolynucleotides or polypeptides, respectively, of the present invention;and, (b) a consensus sequence of at least three homologouspolynucleotides or polypeptides, respectively, of the present invention.The generic sequence of the present invention comprises each species ofpolypeptide or polynucleotide embraced by the generic polypeptide orpolynucleotide sequence, respectively. The individual speciesencompassed by a polynucleotide having an amino acid or nucleic acidconsensus sequence can be used to generate antibodies or produce nucleicacid probes or primers to screen for homologs in other species, genera,families, orders, classes, phyla, or kingdoms. For example, apolynucleotide having a consensus sequence from a gene family of Zeamays can be used to generate antibody or nucleic acid probes or primersto other Gramineae species such as wheat, rice, or sorghum.Alternatively, a polynucleotide having a consensus sequence generatedfrom orthologous genes can be used to identify or isolate orthologs ofother taxa. Typically, a polynucleotide having a consensus sequence willbe at least 9, 10, 15, 20, 25, 30, or 40 amino acids in length, or 20,30, 40, 50, 100, or 150 nucleotides in length. As those of skill in theart are aware, a conservative amino acid substitution can be used foramino acids which differ amongst aligned sequence but are from the sameconservative substitution group as discussed above. Optionally, no morethan 1 or 2 conservative amino acids are substituted for each 10 aminoacid length of consensus sequence.

[0178] Similar sequences used for generation of a consensus or genericsequence include any number and combination of allelic variants of thesame gene, orthologous, or paralogous sequences as provided herein.Optionally, similar sequences used in generating a consensus or genericsequence are identified using the BLAST algorithm's smallest sumprobability (P(N)). Various suppliers of sequence-analysis software arelisted in chapter 7 of Current Protocols in Molecular Biology, F. M.Ausubel et al., Eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (Supplement 30).A polynucleotide sequence is considered similar to a reference sequenceif the smallest sum probability in a comparison of the test nucleic acidto the reference nucleic acid is less than about 0.1, more preferablyless than about 0.01, or 0.001, and most preferably less than about0.0001, or 0.00001. Similar polynucleotides can be aligned and aconsensus or generic sequence generated using multiple sequencealignment software available from a number of commercial suppliers suchas the Genetics Computer Group's (Madison, Wis.) PILEUP software, VectorNTI's (North Bethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.)SEQUENCHER. Conveniently, default parameters of such software can beused to generate consensus or generic sequences.

[0179] Machine Applications

[0180] The present invention provides machines, data structures, andprocesses for modeling or analyzing the polynucleotides and polypeptidesof the present invention.

[0181] A. Machines: Data, Data Structures, Processes, and Functions

[0182] The present invention provides a machine having a memorycomprising: 1) data representing a sequence of a polynucleotide orpolypeptide of the present invention, 2) a data structure which reflectsthe underlying organization and structure of the data and facilitatesprogram access to data elements corresponding to logical sub-componentsof the sequence, 3) processes for effecting the use, analysis, ormodeling of the sequence, and 4) optionally, a function or utility forthe polynucleotide or polypeptide. Thus, the present invention providesa memory for storing data that can be accessed by a computer programmedto implement a process for effecting the use, analyses, or modeling of asequence of a polynucleotide, with the memory comprising datarepresenting the sequence of a polynucleotide of the present invention.

[0183] The machine of the present invention is typically a digitalcomputer. The term “computer” includes one or several desktop orportable computers, computer workstations, servers (including intranetor internet servers), mainframes, and any integrated system comprisingany of the above irrespective of whether the processing, memory, input,or output of the computer is remote or local, as well as any networkinginterconnecting the modules of the computer. The term “computer” isexclusive of computers of the United States Patent and Trademark Officeor the European Patent Office when data representing the sequence ofpolypeptides or polynucleotides of the present invention is used forpatentability searches.

[0184] The present invention contemplates providing as data a sequenceof a polynucleotide of the present invention embodied in a computerreadable medium. As those of skill in the art will be aware, the form ofmemory of a machine of the present invention, or the particularembodiment of the computer readable medium, are not critical elements ofthe invention and can take a variety of forms. The memory of such amachine includes, but is not limited to, ROM, or RAM, or computerreadable media such as, but not limited to, magnetic media such ascomputer disks or hard drives, or media such as CD-ROMs, DVDs, and thelike.

[0185] The present invention further contemplates providing a datastructure that is also contained in memory. The data structure may bedefined by the computer programs that define the processes (see below)or it may be defined by the programming of separate data storage andretrieval programs subroutines, or systems. Thus, the present inventionprovides a memory for storing a data structure that can be accessed by acomputer programmed to implement a process for effecting the use,analysis, or modeling of a sequence of a polynucleotide. The memorycomprises data representing a polynucleotide having the sequence of apolynucleotide of the present invention. The data is stored withinmemory. Further, a data structure, stored within memory, is associatedwith the data reflecting the underlying organization and structure ofthe data to facilitate program access to data elements corresponding tological sub-components of the sequence. The data structure enables thepolynucleotide to be identified and manipulated by such programs.

[0186] In a further embodiment, the present invention provides a datastructure that contains data representing a sequence of a polynucleotideof the present invention stored within a computer readable medium. Thedata structure is organized to reflect the logical structuring of thesequence, so that the sequence is easily analyzed by software programscapable of accessing the data structure. In particular, the datastructures of the present invention organize the reference sequences ofthe present invention in a manner which allows software tools to performa wide variety of analyses using logical elements and sub-elements ofeach sequence.

[0187] An example of such a data structure resembles a layered hashtable, where in one dimension the base content of the sequence isrepresented by a string of elements A, T, C, G and N. The direction fromthe 5′ end to the 3′ end is reflected by the order from the position 0to the position of the length of the string minus one. Such a string,corresponding to a nucleotide sequence of interest, has a certain numberof substrings, each of which is delimited by the string position of its5′ end and the string position of its 3′ end within the parent string.In a second dimension, each substring is associated with or pointed toone or multiple attribute fields. Such attribute fields containannotations to the region on the nucleotide sequence represented by thesubstring.

[0188] For example, a sequence under investigation is 520 bases long andrepresented by a string named SeqTarget. There is a minor groove in the5′ upstream non-coding region from position 12 to 38, which isidentified as a binding site for an enhancer protein HM-A, which in turnwill increase the transcription of the gene represented by SeqTarget.Here, the substring is represented as (12, 38) and has the followingattributes: [upstream uncoded], [minor groove], [HM-A binding] and[increase transcription upon binding by HM-A]. Similarly, other types ofinformation can be stored and structured in this manner, such asinformation related to the whole sequence, e.g., whether the sequence isa full length viral gene, a mammalian house keeping gene or an EST fromclone X, information related to the 3′ down stream non-coding region,e.g., hair pin structure, and information related to various domains ofthe coding region, e.g., Zinc finger.

[0189] This data structure is an open structure and is robust enough toaccommodate newly generated data and acquired knowledge. Such astructure is also a flexible structure. It can be trimmed down to a 1-Dstring to facilitate data mining and analysis steps, such as clustering,repeat-masking, and HMM analysis. Meanwhile, such a data structure alsocan extend the associated attributes into multiple dimensions. Pointerscan be established among the dimensioned attributes when needed tofacilitate data management and processing in a comprehensive genomicsknowledgebase. Furthermore, such a data structure is object-oriented.Polymorphism can be represented by a family or class of sequenceobjects, each of which has an internal structure as discussed above. Thecommon traits are abstracted and assigned to the parent object, whereaseach child object represents a specific variant of the family or class.Such a data structure allows data to be efficiently retrieved, updatedand integrated by the software applications associated with the sequencedatabase and/or knowledgebase.

[0190] The present invention contemplates providing processes foreffecting analysis and modeling, which are described in the followingsection.

[0191] Optionally, the present invention further contemplates that themachine of the present invention will embody in some manner a utility orfunction for the polynucleotide or polypeptide of the present invention.The function or utility of the polynucleotide or polypeptide can be afunction or utility for the sequence data, per se, or of the tangiblematerial. Exemplary function or utilities include the name (perInternational Union of Biochemistry and Molecular Biology rules ofnomenclature) or function of the enzyme or protein represented by thepolynucleotide or polypeptide of the present invention; the metabolicpathway of the protein represented by the polynucleotide or polypeptideof the present invention; the substrate or product or structural role ofthe protein represented by the polynucleotide or polypeptide of thepresent invention; or, the phenotype (e.g., an agronomic orpharmacological trait) affected by modulating expression or activity ofthe protein represented by the polynucleotide or polypeptide of thepresent invention.

[0192] B. Computer Analysis and Modeling

[0193] The present invention provides a process of modeling andanalyzing data representative of a polynucleotide or polypeptidesequence of the present invention. The process comprises enteringsequence data of a polynucleotide or polypeptide of the presentinvention into a machine having a hardware or software sequence modelingand analysis system, developing data structures to facilitate access tothe sequence data, manipulating the data to model or analyze thestructure or activity of the polynucleotide or polypeptide, anddisplaying the results of the modeling or analysis. Thus, the presentinvention provides a process for effecting the use, analysis, ormodeling of a polynucleotide sequence or its derived peptide sequencethrough use of a computer having a memory. The process comprises 1)placing into the memory data representing a polynucleotide having thesequence of a polynucleotide of the present invention, developing withinthe memory a data structure associated with the data and reflecting theunderlying organization and structure of the data to facilitate programaccess to data elements corresponding to logical sub-components of thesequence, 2) programming the computer with a program containinginstructions sufficient to implement the process for effecting the use,analysis, or modeling of the polynucleotide sequence or the peptidesequence, and, 3) executing the program on the computer while grantingthe program access to the data and to the data structure within thememory.

[0194] A variety of modeling and analytic tools are well known in theart and available commercially. Included amongst the modeling/analysistools are methods to: 1) recognize overlapping sequences (e.g., from asequencing project) with a polynucleotide of the present invention andcreate an alignment called a “contig”; 2) identify restriction enzymesites of a polynucleotide of the present invention; 3) identify theproducts of a T1 ribonuclease digestion of a polynucleotide of thepresent invention; 4) identify PCR primers with minimalself-complementarity; 5) compute pairwise distances between sequences inan alignment, reconstruct phylogentic trees using distance methods, andcalculate the degree of divergence of two protein coding regions; 6)identify patterns such as coding regions, terminators, repeats, andother consensus patterns in polynucleotides of the present invention; 7)identify RNA secondary structure; 8) identify sequence motifs,isoelectric point, secondary structure, hydrophobicity, and antigenicityin polypeptides of the present invention; 9) translate polynucleotidesof the present invention and backtranslate polypeptides of the presentinvention; and 10) compare two protein or nucleic acid sequences andidentifying points of similarity or dissimilarity between them.

[0195] The processes for effecting analysis and modeling can be producedindependently or obtained from commercial suppliers. Exemplary analysisand modeling tools are provided in products such as InforMax's(Bethesda, Md.) Vector NTI Suite (Version 5.5), Intelligenetics'(Mountain View, Calif.) PC/Gene program, and Genetics Computer Group's(Madison, Wis.) Wisconsin Package (Version 10.0); these tools, and thefunctions they perform, (as provided and disclosed by the programs andaccompanying literature) are incorporated herein by reference and aredescribed in more detail in section C which follows.

[0196] Thus, in a further embodiment, the present invention provides amachine-readable media containing a computer program and data,comprising a program stored on the media containing instructionssufficient to implement a process for effecting the use, analysis, ormodeling of a representation of a polynucleotide or peptide sequence.The data stored on the media represents a sequence of a polynucleotidehaving the sequence of a polynucleotide of the present invention. Themedia also includes a data structure reflecting the underlyingorganization and structure of the data to facilitate program access todata elements corresponding to logical sub-components of the sequence,the data structure being inherent in the program and in the way in whichthe program organizes and accesses the data.

[0197] C. Homology Searches

[0198] As an example of such a comparative analysis, the presentinvention provides a process of identifying a candidate homologue (i.e.,an ortholog or paralog) of a polynucleotide or polypeptide of thepresent invention. The process comprises entering sequence data of apolynucleotide or polypeptide of the present invention into a machinehaving a hardware or software sequence analysis system, developing datastructures to facilitate access to the sequence data, manipulating thedata to analyze the structure the polynucleotide or polypeptide, anddisplaying the results of the analysis. A candidate homologue hasstatistically significant probability of having the same biologicalfunction (e.g., catalyzes the same reaction, binds to homologousproteins/nucleic acids, has a similar structural role) as the referencesequence to which it is compared. Accordingly, the polynucleotides andpolypeptides of the present invention have utility in identifyinghomologs in animals or other plant species, particularly those in thefamily Gramineae such as, but not limited to, sorghum, wheat, or rice.

[0199] The process of the present invention comprises obtaining datarepresenting a polynucleotide or polypeptide test sequence. Testsequences can be obtained from a nucleic acid of an animal or plant.Test sequences can be obtained directly or indirectly from sequencedatabases including, but not limited to, those such as: GenBank, EMBL,GenSeq, SWISS-PROT, or those available on-line via the UK Human GenomeMapping Project (HGMP) GenomeWeb. In some embodiments the test sequenceis obtained from a plant species other than maize whose function isuncertain but will be compared to the test sequence to determinesequence similarity or sequence identity. The test sequence data isentered into a machine, such as a computer, containing: i) datarepresenting a reference sequence and, ii) a hardware or softwaresequence comparison system to compare the reference and test sequencefor sequence similarity or identity.

[0200] Exemplary sequence comparison systems are provided for insequence analysis software such as those provided by the GeneticsComputer Group (Madison, Wis.) or InforMax (Bethesda, Md.), orIntelligenetics (Mountain View, Calif.). Optionally, sequence comparisonis established using the BLAST or GAP suite of programs. Generally, asmallest sum probability value (P(N)) of less than 0.1, oralternatively, less than 0.01, 0.001, 0.0001, or 0.00001 using the BLAST2.0 suite of algorithms under default parameters identifies the testsequence as a candidate homologue (i.e., an allele, ortholog, orparalog) of the reference sequence. Those of skill in the art willrecognize that a candidate homologue has an increased statisticalprobability of having the same or similar function as the gene/proteinrepresented by the test sequence.

[0201] The reference sequence can be the sequence of a polypeptide or apolynucleotide of the present invention. The reference or test sequenceis each optionally at least 25 amino acids or at least 100 nucleotidesin length. The length of the reference or test sequences can be thelength of the polynucleotide or polypeptide described, respectively,above in the sections entitled “Nucleic Acids”(particularly section(g)), and “Proteins”. As those of skill in the art are aware, thegreater the sequence identity/similarity between a reference sequence ofknown function and a test sequence, the greater the probability that thetest sequence will have the same or similar function as the referencesequence. The results of the comparison between the test and referencesequences are outputted (e.g., displayed, printed, recorded) via any oneof a number of output devices and/or media (e.g., computer monitor, hardcopy, or computer readable medium).

[0202] Detection of Nucleic Acids

[0203] The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of containing a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of maize. In someembodiments, a cognate gene of a polynucleotide of the present inventionor portion thereof can be amplified prior to the step of contacting thenucleic acid sample with a polynucleotide of the present invention. Thenucleic acid sample is contacted with the polynucleotide to form ahybridization complex. The polynucleotide hybridizes under stringentconditions to a gene encoding a polypeptide of the present invention.Formation of the hybridization complex is used to detect a gene encodinga polypeptide of the present invention in the nucleic acid sample. Thoseof skill will appreciate that an isolated nucleic acid comprising apolynucleotide of the present invention should lack cross-hybridizingsequences in common with non-target genes that would yield a falsepositive result. Detection of the hybridization complex can be achievedusing any number of well known methods. For example, the nucleic acidsample, or a portion thereof, may be assayed by hybridization formatsincluding but not limited to, solution phase, solid phase, mixed phase,or in situ hybridization assays.

[0204] Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include biotinfor staining with labeled streptavidin conjugate, magnetic beads,fluorescent dyes, radiolabels, enzymes, and colorimetric labels. Otherlabels include ligands which bind to antibodies labeled withfluorophores, chemiluminescent agents, and enzymes. Labeling the nucleicacids of the present invention is readily achieved such as by the use oflabeled PCR primers.

[0205] Although the present invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

EXAMPLE 1

[0206] This example describes the construction of a cDNA library.

[0207] Total RNA can be isolated from maize tissues with TRIzol Reagent(Life Technology Inc. Gaithersburg, Md.) using a modification of theguanidine isothiocyanate/acid-phenol procedure described by Chomczynskiand Sacchi (Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156(1987)). In brief, plant tissue samples is pulverized in liquid nitrogenbefore the addition of the TRIzol Reagent, and then further homogenizedwith a mortar and pestle. Addition of chloroform followed bycentrifugation is conducted for separation of an aqueous phase and anorganic phase. The total RNA is recovered by precipitation withisopropyl alcohol from the aqueous phase.

[0208] The selection of poly(A)+ RNA from total RNA can be performedusing PolyATact system (Promega Corporation. Madison, Wis.).Biotinylated oligo(dT) primers are used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids are captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA is thenwashed at high stringency conditions and eluted by RNase-free deionizedwater.

[0209] cDNA synthesis and construction of unidirectional cDNA librariescan be accomplished using the SuperScript Plasmid System (LifeTechnology Inc. Gaithersburg, Md.). The first strand of cDNA issynthesized by priming an oligo(dT) primer containing a Not I site. Thereaction is catalyzed by SuperScript Reverse Transcriptase II at 45° C.The second strand of cDNA is labeled with alpha-³²P-dCTP and a portionof the reaction analyzed by agarose gel electrophoresis to determinecDNA sizes. cDNA molecules smaller than 500 base pairs and unligatedadapters are removed by Sephacryl-S400 chromatography. The selected cDNAmolecules are ligated into pSPORT1 vector in between of Not I and Sal Isites.

[0210] Alternatively, cDNA libraries can be prepared by any one of manymethods available. For example, the cDNAs may be introduced into plasmidvectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectorsaccording to the manufacturer's protocol (Stratagene Cloning Systems, LaJolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmidlibraries according to the protocol provided by Stratagene. Uponconversion, cDNA inserts will be contained in the plasmid vectorpBluescript. In addition, the cDNAs may be introduced directly intoprecut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (NewEngland Biolabs), followed by transfection into DH10B cells according tothe manufacturer's protocol (GIBCO BRL Products). Once the cDNA insertsare in plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

EXAMPLE 2

[0211] This method describes construction of a full-length enriched cDNAlibrary.

[0212] An enriched full-length cDNA library can be constructed using oneof two variations of the method of Carninci et al. Genomics 37:327-336,1996. These variations are based on chemical introduction of abiotin group into the diol residue of the 5′0 cap structure ofeukaryotic mRNA to select full-length first strand cDNA. The selectionoccurs by trapping the biotin residue at the cap sites usingstreptavidin-coated magnetic beads followed by RNase I treatment toeliminate incompletely synthesized cDNAs. Second strand cDNA issynthesized using established procedures such as those provided in LifeTechnologies' (Rockville, Md.) “SuperScript Plasmid System for cDNASynthesis and Plasmid Cloning” kit. Libraries made by this method havebeen shown to contain 50% to 70% full-length cDNAs.

[0213] The first strand synthesis methods are detailed below. Anasterisk denotes that the reagent was obtained from Life Technologies,Inc.

[0214] A. First Strand cDNA Synthesis Method 1 (with Trehalose) mRNA (10ug) 25 μl *Not I primer (5 ug) 10 μl *5x 1^(st) strand buffer 43 μl *0.1m DTT 20 μl *dNTP mix 10 mm 10 μl BSA 10 ug/μl 1 μl Trehalose(saturated) 59.2 μl RNase inhibitor (Promega) 1.8 μl *Superscript II RT200 u/μl 20 μl 100% glycerol 18 μl Water 7 μl

[0215] The mRNA and Not I primer are mixed and denatured at 65° C. for10 min. They are then chilled on ice and other components added to thetube. Incubation is at 45° C. for 2 min. Twenty microliters of RT(reverse transcriptase) is added to the reaction and start program onthe thermocycler (MJ Research, Waltham, Ma.): Step 1 45° C. 10 min Step2 45° C. −0.3° C./cycle, 2 seconds/cycle Step 3 go to 2 for 33 cyclesStep 4 35° C. 5 min Step 5 45° C. 5 min Step 6 45° C. 0.2° C./cycle, 1sec/cycle Step 7 go to 7 for 49 cycles Step 8 55° C. 0.1° C./cycle, 12sec/cycle Step 9 go to 8 for 49 cycles Step 10 55° C. 2 min Step 11 60°C. 2 min Step 12 go to 11 for 9 times Step 13 4° C. forever Step 14 end

[0216] B. First Strand cDNA Synthesis Method 2 mRNA (10 μg) 25 μl water30 μl *Not I adapter primer (5 μg) 10 μl 65° C. for 10 min, chill onice, then add following reagents, *5x first buffer 20 μl *0.1 M DTT 10μl *10 mM dNTP mix  5 μl

[0217] Incubate at 45° C. for 2 min, then add 10 μl of *Superscript IIRT (200 u/μl), start the following program: Step 1 45° C. for 6 sec,−0.1° C./cycle Step 2 go to 1 for 99 additional cycles Step 3 35° C. for5 min Step 4 45° C. for 60 min Step 5 50° C. for 10 min Step 6 4° C.forever Step 7 end

[0218] After the 1^(st) strand cDNA synthesis, the DNA is extracted byphenol according to standard procedures, and then precipitated in NaOAcand ethanol, and stored in −200° C. ps C. Oxidization of the Diol Groupof mRNA for Biotin Labeling

[0219] First strand cDNA is spun down and washed once with 70% EtOH. Thepellet resuspended in 23.2 μl of DEPC treated water and put on ice.Prepare 100 mM of NalO4 freshly, and then add the following reagents:mRNA:1^(st) cDNA (start with 20 μg mRNA) 46.4 μl 100 mM NaIO4 (freshlymade)  2.5 μl NaOAc 3M pH 4.5  1.1 μl

[0220] To make 100 mM NalO4, use 21.39 μg of NalO4 for 1 μl water.

[0221] Wrap the tube in a foil and incubate on ice for 45 min.

[0222] After the incubation, the reaction is then precipitated in: 5 MNaCl  10 μl 20% SDS 0.5 μl isopropanol  61 μl

[0223] Incubate on ice for at least 30 min, then spin it down at maxspeed at 4° C. for 30 min and wash once with 70% ethanol and then 80%EtOH.

[0224] D. Biotinylation of the mRNA Diol Group

[0225] Resuspend the DNA in 110 μl DEPC treated water, then add thefollowing reagents: 20% SDS  5 μl 2 M NaOAc pH 6.1  5 μl 10 mm biotinhydrazide (freshly made) 300 μl

[0226] Wrap in a foil and incubate at room temperature overnight.

[0227] E. RNase I Treatment

[0228] Precipitate DNA in: 5 M NaCl 10 μl 2 M NaOAc pH 6.1 75 μlbiotinylated mRNA:cDNA 420 μl 100% EtOH (2.5 Vol) 1262.5 μl

[0229] (Perform this precipitation in two tubes and split the 420 μl ofDNA into 210 μl each, add 5 μl of 5M NaCl, 37.5 μl of 2M NaOAc pH 6.1,and 631.25 μl of 100% EtOH). Store at −20° C. for at least 30 min. Spinthe DNA down at 4° C. at maximal speed for 30 min. and wash with 80%EtOH twice, then dissolve DNA in 70 μl RNase free water. Pool two tubesand end up with 140 μl.

[0230] Add the following reagents: RNase One 10 U/μl  40 μl 1^(st)cDNA:RNA 140 μl 10X buffer  20 μl

[0231] Incubate at 37° C. for 15 min.

[0232] Add 5 μl of 40 μg/μl yeast tRNA to each sample for capturing.

[0233] F. Full Length 1^(st) cDNA Capturing

[0234] Blocking the beads with yeast tRNA: Beads 1 ml Yeast tRNA 40μg/μl 5 μl

[0235] Incubate on ice for 30 min with mixing, wash 3 times with 1 ml of2M NaCl, 50 mmEDTA, pH 8.0.

[0236] Resuspend the beads in 800 μl of 2M NaCl , 50 mm EDTA, pH 8.0,add RNase I treated sample 200 μl, and incubate the reaction for 30 minat room temperature. Capture the beads using the magnetic stand, savethe supernatant, and start following washes:

[0237] 2 washes with 2M NaCl, 50 mm EDTA, pH 8.0, 1 ml each time,

[0238] 1 wash with 0.4% SDS, 50 μg/ml tRNA,

[0239] 1 wash with 10 mm Tris-Cl pH 7.5, 0.2 mm EDTA, 10 mm NaCl, 20%glycerol,

[0240] 1 wash with 50 μg/ml tRNA,

[0241] 1 wash with 1^(st) cDNA buffer

[0242] G. Second Strand cDNA Synthesis

[0243] Resuspend the beads in: *5X first buffer 8 μl *0.1 mM DTT 4 μl*10 mm dNTP mix 8 μl *5X 2nd buffer 60 μl  *E. coli Ligase 10 U/μl 2 μl*E. coli DNA polymerase 10 U/μl 8 μl *E. coli RNaseH 2 U/μl 2 μl P32dCTP 10 μci/μl 2 μl Or water up to 300 μl 208 μl 

[0244] Incubate at 16° C. for 2 hr with mixing the reaction in every 30min.

[0245] Add 4 μl of T4 DNA polymerase and incubate for additional 5 minat 16° C.

[0246] Elute 2^(nd) cDNA from the beads.

[0247] Use a magnetic stand to separate the 2^(nd) cDNA from the beads,then resuspend the beads in 200 μl of water, and then separate again,pool the samples (about 500 μl), Add 200 μl of water to the beads, then200 μl of phenol:chloroform, vortex, and spin to separate the samplewith phenol.

[0248] Pool the DNA together (about 700 μl) and use phenol to clean theDNA again, DNA is then precipitated in 2 μg of glycogen and 0.5 vol of7.5M NH4OAc and 2 vol of 100% EtOH. Precipitate overnight. Spin down thepellet and wash with 70% EtOH, air-dry the pellet. DNA 250 μl DNA 200 μl7.5 M NH4OAc 125 μl 7.5 M NH4OAc 100 μl 100% EtOH 750 μl 100% EtOH 600μl glycogen 1 μg/μl  2 μl glycogen 1 μg/μl  2 μl

[0249] H. Sal I Adapter Ligation

[0250] Resuspend the pellet in 26 μl of water and use 1 μl for TAE gel.

[0251] Set up reaction as following: 2^(nd) strand cDNA 25 μl *5X T4 DNAligase buffer 10 μl *Sal I adapters 10 μl *T4 DNA ligase  5 μl

[0252] Mix gently, incubate the reaction at 16° C. overnight.

[0253] Add 2 μl of ligase second day and incubate at room temperaturefor 2 hrs (optional).

[0254] Add 50 μl water to the reaction and use 100 μl of phenol to cleanthe DNA, 90 μl of the upper phase is transferred into a new tube, andprecipitate in: Glycogen 1 μg/μl  2 μl Upper phase DNA  90 μl 7.5 MNH4OAc  50 μl 100% EtOH 300 μl

[0255] precipitate at −20° C. overnight

[0256] Spin down the pellet at 4° C. and wash in 70% EtOH, dry thepellet.

[0257] I. Not I Digestion 2^(nd) cDNA 41 μl *Reaction 3 buffer  5 μl*Not I 15 u/μl  4 μl

[0258] Mix gently and incubate the reaction at 37° C. for 2 hr.

[0259] Add 50 μl of water and 100 μl of phenol, vortex, and take 90 μlof the upper phase to a new tube, then add 50 μl of NH40Ac and 300 μl ofEtOH. Precipitate overnight at −20° C.

[0260] Cloning, ligation, and transformation are performed per theSuperscript cDNA synthesis kit.

EXAMPLE 3

[0261] This example describes cDNA sequencing and library subtraction.

[0262] Individual colonies can be picked and DNA prepared either by PCRwith M13 forward primers and M13 reverse primers, or by plasmidisolation. cDNA clones can be sequenced using M13 reverse primers.

[0263] cDNA libraries are plated out on 22×22 cm² agar plate at densityof about 3,000 colonies per plate. The plates are incubated in a 37° C.incubator for 12-24 hours. Colonies are picked into 384-well plates by arobot colony picker, Q-bot (GENETIX Limited). These plates are incubatedovernight at 37° C. Once sufficient colonies are picked, they are pinnedonto 22×22 cm² nylon membranes using Q-bot. Each membrane holds 9,216 or36,864 colonies. These membranes are placed onto an agar plate with anappropriate antibiotic. The plates are incubated at 37° C. overnight.

[0264] After colonies are recovered on the second day, these filters areplaced on filter paper prewetted with denaturing solution for fourminutes, then incubated on top of a boiling water bath for an additionalfour minutes. The filters are then placed on filter paper prewetted withneutralizing solution for four minutes. After excess solution is removedby placing the filters on dry filter papers for one minute, the colonyside of the filters is placed into Proteinase K solution, incubated at37° C. for 40-50 minutes. The filters are placed on dry filter papers todry overnight. DNA is then cross-linked to nylon membrane by UV lighttreatment.

[0265] Colony hybridization is conducted as described by Sambrook,J.,Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A laboratoryManual, 2^(nd) Edition). The following probes can be used in colonyhybridization:

[0266] 1. First strand cDNA from the same tissue as the library was madefrom to remove the most redundant clones.

[0267] 2. 48-192 most redundant cDNA clones from the same library basedon previous sequencing data.

[0268] 3. 192 most redundant cDNA clones in the entire maize sequencedatabase.

[0269] 4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAAAAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.

[0270] 5. cDNA clones derived from rRNA.

[0271] The image of the autoradiography is scanned into computer and thesignal intensity and cold colony addresses of each colony is analyzed.Re-arraying of cold-colonies from 384 well plates to 96 well plates isconducted using Q-bot.

EXAMPLE 4

[0272] This example describes identification of the gene from a computerhomology search.

[0273] Gene identities can be determined by conducting BLAST (BasicLocal Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol.Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches underdefault parameters for similarity to sequences contained in the BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences are analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm. The DNA sequences are translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993))provided by the NCBI. In some cases, the sequencing data from two ormore clones containing overlapping segments of DNA are used to constructcontiguous DNA sequences.

[0274] Sequence alignments and percent identity calculations can beperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences can be performed using the Clustal method of alignment(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method are KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

EXAMPLE 5

[0275] This example describes expression of transgenes in monocot cells.

[0276] A transgene comprising a cDNA encoding the instant polypeptidesin sense orientation with respect to the maize 27 kD zein promoter thatis located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes Ncol and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcol-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SaII-Ncol promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SaII fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (SequenaseDNA Sequencing Kit; U. S. Biochemical). The resulting plasmid constructwould comprise a transgene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

[0277] The transgene described above can then be introduced into maizecells by the following procedure. Immature maize embryos can bedissected from developing caryopses derived from crosses of the inbredmaize lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0278] The plasmid, p35S/Ac (Hoechst Ag, Frankfurt, Germany) orequivalent may be used in transformation experiments in order to providefor a selectable marker. This plasmid contains the Pat gene (seeEuropean Patent Publication 0 242 236) which encodes phosphinothricinacetyl transferase (PAT). The enzyme PAT confers resistance toherbicidal glutamine synthetase inhibitors such as phosphinothricin. Thepat gene in p35S/Ac is under the control of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) andthe 3′ region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agrobacterium tumefaciens.

[0279] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton flying disc (Bio-Rad Labs). The particles are thenaccelerated into the maize tissue with a Biolistic PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0280] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covers a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0281] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0282] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

EXAMPLE 6

[0283] This example describes expression of transgenes in dicot cells.

[0284] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by Hind III sites.

[0285] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0286] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0287] Soybean embryogenic suspension cultures can maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0288] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0289] A selectable marker gene which can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al.(1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0290] To 50 μL of a 60 μg/mL 1 μmgold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

[0291] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0292] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

EXAMPLE 7

[0293] This example describes expression of a transgene in microbialcells.

[0294] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0295] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly, Ma.).The fragment containing the ligated adapters can be purified from theexcess adapters using low melting agarose as described above. The vectorpBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) anddeproteinized with phenol/chloroform as described above. The preparedvector pBT430 and fragment can then be ligated at 16° C. for 15 hoursfollowed by transformation into DH5 electrocompetent cells (GIBCO BRL).Transformants can be selected on agar plates containing LB media and 100μg/mL ampicillin. Transformants containing the gene encoding the instantpolypeptides are then screened for the correct orientation with respectto the T7 promoter by restriction enzyme analysis.

[0296] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol.Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 250. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One microgramof protein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

EXAMPLE 8

[0297] This example describes a procedure to identify plants containingMu inserted into genes of interest and a strategy to identify thefunction of those genes. This example is based on work with the CQRAD17gene, described in U.S. patent application Ser. No. 09/371,383 anddisclosed therein as Seq. ID No. 25, which is a member of the same genefamily as SEQ ID Nos. 1 and 5 of the present application. One of skillin the art could readily conceive of use of this procedure with thesequences disclosed in the current application.

[0298] The Trait Utility System for Corn (TUSC) is a method that employsgenetic and molecular techniques to facilitate the study of genefunction in maize. Studying gene function implies that the gene'ssequence is already known, thus the method works in reverse: fromsequence to phenotype. This kind of application is referred to as“reverse genetics”, which contrasts with “forward” methods that aredesigned to identify and isolate the gene(s) responsible for aparticular trait (phenotype).

[0299] Pioneer Hi-Bred International, Inc., has a proprietary collectionof maize genomic DNA from approximately 42,000 individual F₁ plants(Reverse genetics for maize, Meeley, R. and Briggs, S.,1995, MaizeGenet. Coop. Newslett. 69:67, 82). The genome of each of theseindividuals contains multiple copies of the transposable element family,Mutator (Mu). The Mu family is highly mutagenic; in the presence of theactive element Mu-DR, these elements transpose throughout the genome,inserting into genic regions, and often disrupting gene function. Bycollecting genomic DNA from a large number (42,000) of individuals,Pioneer has assembled a library of the mutagenized maize genome.

[0300] Mu insertion events are predominantly heterozygous; given therecessive nature of most insertional mutations, the F₁ plants appearwild-type. Each of the F₁ plants is selfed to produce F₂ seed, which iscollected. In generating the F₂ progeny, insertional mutations segregatein a Mendelian fashion so are useful for investigating a mutant allele'seffect on the phenotype. The TUSC system has been successfully used by anumber of laboratories to identify the function of a variety of genes(Cloning and characterization of the maize An1 gene, Bensen, R. J., etal., 1995, Plant Cell 7:75-84; Diversification of C-function activity inmaize flower development, Mena, M., et al., 1996, Science 274:1537-1540;Analysis of a chemical plant defense mechanism in grasses, Frey, M., etal., 1997, Science 277:696-699;The control of maize spikelet meristemfate by the APETALA2-like gene Indeterminate spikelet 1, Chuck, G.,Meeley, R. B., and Hake, S., 1998, Genes & Development 12:1145-1154; ASecY homologue is required for the elaboration of the chloroplastthylakoid membrane and for normal chloroplast gene expression, Roy, L.M. and Barkan, A., 1998, J. Cell Biol. 141:1-11).

[0301] PCR Screening for Mu insertions in CQRAD17:

[0302] Two primers were designed from within the CQRAD17 cDNA anddesignated as gene-specific primers (GSPs): Forward primer (GSP1): 5′-TGC TGA TAT CGA GAA GGC CGG AAT CGT -3′ Reverse primer (GSP2): 5′- CTCCCC ACC AGA CCC TTG AGG -3′ Mu TIR primer: 5′- AGA GAA GCC AAC GCC AWCGCC TCY ATT TCG TC -3′

[0303] Pickoligo was used to select primers for PCR. This programchooses the Tm according to the following equation:

Tm=[((GC*3+AT*2)*37−562)/length]−5

[0304] PCR reactions were run with an annealing temperature of 62 □C anda thermocycling profile as follows: 94° C. — 2′ (initial denaturation)94° C. — 30″-1′ 35 cycles {close oversize brace} 62° C. — 30″-2′ 72° C.— 1-3′ 72° C. — 5′ (final extension)

[0305] Gel electrophoresis of the PCR products confirmed that there wasno false priming in single primer reactions and that only one fragmentwas amplified in paired GSP reactions.

[0306] The genomic DNA from 42,000 plants, combined into pools of 48plants each, was subjected to PCR with either GSP1 or GSP2 and Mu TIR.The pools that were confirmed to be positive by dot-blot hybridizationusing CQRAD17 cDNA as a probe were subjected to gel-blot analysis inorder to determine the size of fragments amplified. The pools in whichclean fragments were identified were subjected to further analysis toidentify the individual plants within those pools that contained Muinsertion(s).

[0307] Seed from F₁ plants identified in this manner was planted in thefield. Leaf discs from twenty plants in each F₂ row were collected andgenomic DNA was isolated. The same twenty plants were selfed and the F₃seed saved. Pooled DNA (from 20 plants) from each of twelve rows wassubjected to PCR using GSP1 or GSP2 and Mu TIR primer as mentionedabove. Three pools identified to contain Mu insertions were subjected toindividual plant analysis and homozygotes identified. The Mu insertionsites with the surrounding signature sequences are identified below:Allele 1: TCTTCACCA-Mu-GGTCCTTCG Allele 2: GTCGAAATT-Mu-TTCTTCAGC Allele3: GCTCACGGG-Mu-GAAGTTTAT

[0308] All three insertions are within 200 nucleotides of each other inthe open reading frame, suggesting that this region in the gene mightrepresent a hot spot for Mu insertion. One of the insertions, allele 3,is in the region predicted to code for a transmembrane domain. Each ofthese insertions is expected to inactivate the gene.

[0309] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications, andcomputer programs cited herein are hereby incorporated by reference.

1 22 1 2830 DNA Zea mays CDS (3)...(2468) misc_feature (1)...(2830) n =A,T,C or G 1 ta cct cta agt cgc ata gtt ccg ata tct cca aac gag ctt aacctt 47 Pro Leu Ser Arg Ile Val Pro Ile Ser Pro Asn Glu Leu Asn Leu 1 510 15 tat cgg atc gtg att gtt ctc cgg ctt atc atc cta tgt ttc ttc ttt 95Tyr Arg Ile Val Ile Val Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe 20 25 30caa tat cgt ata act cat cca gtg gaa gat gct tat ggg ttg tgg ctt 143 GlnTyr Arg Ile Thr His Pro Val Glu Asp Ala Tyr Gly Leu Trp Leu 35 40 45 gtatct gtt att tgt gaa gtt tgg ttt gcc ttg tct tgg ctt cta gat 191 Val SerVal Ile Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp 50 55 60 cag ttccca aag tgg tat cct atc aac cgt gaa act tac ctc gat aga 239 Gln Phe ProLys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg 65 70 75 ctt gca ttgaga tat gat agg gag ggt gag cca tcc cag ttg gct cca 287 Leu Ala Leu ArgTyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro 80 85 90 95 atc gat gtcttt gtt agt aca gtg gat cca ctt aag gaa cct cct cta 335 Ile Asp Val PheVal Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu 100 105 110 att act ggcaac act gtc ctg tcc att ctt gct gtg gat tac cct gtt 383 Ile Thr Gly AsnThr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val 115 120 125 gac aaa gtatca tgt tat gtt tct gat gac ggt tca gct atg ttg act 431 Asp Lys Val SerCys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr 130 135 140 ttt gaa gcgcta tct gaa acc gca gag ttt gca agg aaa tgg gtt ccc 479 Phe Glu Ala LeuSer Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro 145 150 155 ttt tgc aagaaa cac aat att gaa cct agg gct cca gag ttt tac ttt 527 Phe Cys Lys LysHis Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe 160 165 170 175 gct cgaaag ata gat tac cta aag gac aaa ata caa cct tct ttt gtg 575 Ala Arg LysIle Asp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val 180 185 190 aaa gaaagg cgg gct atg aag agg gag tgt gaa gag ttc aaa gta cgg 623 Lys Glu ArgArg Ala Met Lys Arg Glu Cys Glu Glu Phe Lys Val Arg 195 200 205 atc gatgcc ctt gtt gca aaa gcg caa aaa ata cct gag gag ggc tgg 671 Ile Asp AlaLeu Val Ala Lys Ala Gln Lys Ile Pro Glu Glu Gly Trp 210 215 220 acc atggct gat ggc act cct tgg cct ggg aat aac cct aga gat cat 719 Thr Met AlaAsp Gly Thr Pro Trp Pro Gly Asn Asn Pro Arg Asp His 225 230 235 cca ggaatg atc caa gta ttc ttg ggc cac agt ggt ggg ctt gac acg 767 Pro Gly MetIle Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr 240 245 250 255 gatggg aat gag ttg cca cgg ctt gtt tat gtt tct cgt gaa aag agg 815 Asp GlyAsn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg 260 265 270 ccaggc ttc cag cac cac aag aag gct ggt gcc atg aat gct ttg att 863 Pro GlyPhe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile 275 280 285 cgcgta tca gct gtc ctg acg aat ggt gct tat ctt ctt aat gtg gat 911 Arg ValSer Ala Val Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp 290 295 300 tgtgat cac tac ttc aat agc agc aaa gct ctt aga gag gct atg tgt 959 Cys AspHis Tyr Phe Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys 305 310 315 ttcatg atg gat cca gca cta gga agg aaa act tgc tat gtt cag ttt 1007 Phe MetMet Asp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe 320 325 330 335cca caa aga ttt gat ggt ata gac ttg cat gat cga tat gca aac cgg 1055 ProGln Arg Phe Asp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg 340 345 350aac att gtc ttc ttt gat att aat atg aag ggt cta gat ggc att caa 1103 AsnIle Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln 355 360 365gga cct gtt tat gtg gga aca gga tgc tgt ttc aat agg cag gcc ttg 1151 GlyPro Val Tyr Val Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu 370 375 380tat ggc tat gat cct gta ttg aca gaa gct gat ttg gag cct aac att 1199 TyrGly Tyr Asp Pro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile 385 390 395atc att aaa agt tgc tgt ggc gga aga aaa aag aag gac aag agc tat 1247 IleIle Lys Ser Cys Cys Gly Gly Arg Lys Lys Lys Asp Lys Ser Tyr 400 405 410415 att gat tcc aaa aac cgt gat atg aag aga aca gaa tct tcg gct ccc 1295Ile Asp Ser Lys Asn Arg Asp Met Lys Arg Thr Glu Ser Ser Ala Pro 420 425430 atc ttc aac atg gaa gat ata gaa gag gga ttt gaa ggt tac gag gat 1343Ile Phe Asn Met Glu Asp Ile Glu Glu Gly Phe Glu Gly Tyr Glu Asp 435 440445 gaa agg tca ctg ctt atg tct cag aag agc ttg gag aaa cgc ttt ggc 1391Glu Arg Ser Leu Leu Met Ser Gln Lys Ser Leu Glu Lys Arg Phe Gly 450 455460 cag tct cca att ttt att gca tcc acc ttt atg act caa ggt ggc ata 1439Gln Ser Pro Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile 465 470475 ccc cct tca aca aac cca ggt tcc ctg cta aag gaa gct ata cat gtc 1487Pro Pro Ser Thr Asn Pro Gly Ser Leu Leu Lys Glu Ala Ile His Val 480 485490 495 att agt tgt gga tat gag gat aaa aca gaa tgg ggg aaa gag atc gga1535 Ile Ser Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly 500505 510 tgg ata tat ggc tct gtt act gaa gat att tta act ggt ttc aag atg1583 Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met 515520 525 cat gca aga ggt tgg ata tcc atc tac tgc atg cca ctt cgg cct tgc1631 His Ala Arg Gly Trp Ile Ser Ile Tyr Cys Met Pro Leu Arg Pro Cys 530535 540 ttc aag ggt tct gct cca att aat ctt tct gat cgt ctc aac caa gtg1679 Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val 545550 555 tta cgc tgg gct ctt ggt tca gtt gaa att cta ctt agc aga cac tgt1727 Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys 560565 570 575 cct atc tgg tat ggt tac aat gga agg cta aag ctt ctg gag agactg 1775 Pro Ile Trp Tyr Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu580 585 590 gca tac atc aac acc att gtt tat cca att aca tct atc cca ctagta 1823 Ala Tyr Ile Asn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Val595 600 605 gca tac tgc gtc ctt cct gct atc tgt tta ctc acc aac aaa tttatt 1871 Ala Tyr Cys Val Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile610 615 620 att cct gcg att agc aat tat gct ggg gcg ttc ttc atc ctg cttttt 1919 Ile Pro Ala Ile Ser Asn Tyr Ala Gly Ala Phe Phe Ile Leu Leu Phe625 630 635 gct tcc atc ttc gcc act ggt att ttg gag ctt cga tgg agt ggtgtt 1967 Ala Ser Ile Phe Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val640 645 650 655 ggc att gag gat tgg tgg aga aat gag cag ttt tgg gtc attggt ggc 2015 Gly Ile Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile GlyGly 660 665 670 acc tct gca cat ctc ttt gct gtg ttc caa ggt ctc tta aaagtg cta 2063 Thr Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys ValLeu 675 680 685 gca ggg atc gac aca aac ttc acg gtc aca tca aag gca accgat gat 2111 Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Thr AspAsp 690 695 700 gat ggt gat ttt gct gag ctg tat gtg ttc aag tgg aca actctt ctg 2159 Asp Gly Asp Phe Ala Glu Leu Tyr Val Phe Lys Trp Thr Thr LeuLeu 705 710 715 atc ccc ccc acc act gtg ctt gtg att aac ctg gtt ggt atagtg gct 2207 Ile Pro Pro Thr Thr Val Leu Val Ile Asn Leu Val Gly Ile ValAla 720 725 730 735 gga gtg tcg tat gct atc aac agt ggc tac caa tca tggggt cca cta 2255 Gly Val Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp GlyPro Leu 740 745 750 ttc ggg aag ctg ttc ttt gca atc tgg gtg atc ctc cacctc tac cct 2303 Phe Gly Lys Leu Phe Phe Ala Ile Trp Val Ile Leu His LeuTyr Pro 755 760 765 ttc ctg aag ggt ctc atg ggg aag cag aac cgc aca ccgacc atc gtc 2351 Phe Leu Lys Gly Leu Met Gly Lys Gln Asn Arg Thr Pro ThrIle Val 770 775 780 atc gtt tgg tcc gtc ctt ctt gct tcc ata ttc tcg ctgctg tgg gtg 2399 Ile Val Trp Ser Val Leu Leu Ala Ser Ile Phe Ser Leu LeuTrp Val 785 790 795 aag atc gac ccc ttc ata tcc cct acc cag aag gct ctttcc cgt ggg 2447 Lys Ile Asp Pro Phe Ile Ser Pro Thr Gln Lys Ala Leu SerArg Gly 800 805 810 815 cag tgt ggt gta aac tgc tga aatgatccgaactgcctgct gaataacatt 2498 Gln Cys Gly Val Asn Cys * 820 gctccggcacaatcatgatc taccccttcg tgtaaatacc agaggttagg caagactttt 2558 cttggtaggtggcgaagatg tgtcgtttaa gttcactcta ctgcatttgg ggtgggcagc 2618 atgaaactttgtcaacttat gtcgtgctac ttatttgtag ctaagtagca gtaagtagtg 2678 cctgtttcatgttgactgtc gtgactacct gttcaccgtg ggctctggac tgtcgtgatg 2738 taacctgtatgttggaactt caagtactga ttgagctgtt tggtcaatga cattgaggga 2798 ttctctctctngaaattaan acaaantngg nt 2830 2 821 PRT Zea mays 2 Pro Leu Ser Arg IleVal Pro Ile Ser Pro Asn Glu Leu Asn Leu Tyr 1 5 10 15 Arg Ile Val IleVal Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln 20 25 30 Tyr Arg Ile ThrHis Pro Val Glu Asp Ala Tyr Gly Leu Trp Leu Val 35 40 45 Ser Val Ile CysGlu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln 50 55 60 Phe Pro Lys TrpTyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu 65 70 75 80 Ala Leu ArgTyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile 85 90 95 Asp Val PheVal Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile 100 105 110 Thr GlyAsn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp 115 120 125 LysVal Ser Cys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe 130 135 140Glu Ala Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe 145 150155 160 Cys Lys Lys His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala165 170 175 Arg Lys Ile Asp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe ValLys 180 185 190 Glu Arg Arg Ala Met Lys Arg Glu Cys Glu Glu Phe Lys ValArg Ile 195 200 205 Asp Ala Leu Val Ala Lys Ala Gln Lys Ile Pro Glu GluGly Trp Thr 210 215 220 Met Ala Asp Gly Thr Pro Trp Pro Gly Asn Asn ProArg Asp His Pro 225 230 235 240 Gly Met Ile Gln Val Phe Leu Gly His SerGly Gly Leu Asp Thr Asp 245 250 255 Gly Asn Glu Leu Pro Arg Leu Val TyrVal Ser Arg Glu Lys Arg Pro 260 265 270 Gly Phe Gln His His Lys Lys AlaGly Ala Met Asn Ala Leu Ile Arg 275 280 285 Val Ser Ala Val Leu Thr AsnGly Ala Tyr Leu Leu Asn Val Asp Cys 290 295 300 Asp His Tyr Phe Asn SerSer Lys Ala Leu Arg Glu Ala Met Cys Phe 305 310 315 320 Met Met Asp ProAla Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro 325 330 335 Gln Arg PheAsp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn 340 345 350 Ile ValPhe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly 355 360 365 ProVal Tyr Val Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr 370 375 380Gly Tyr Asp Pro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Ile 385 390395 400 Ile Lys Ser Cys Cys Gly Gly Arg Lys Lys Lys Asp Lys Ser Tyr Ile405 410 415 Asp Ser Lys Asn Arg Asp Met Lys Arg Thr Glu Ser Ser Ala ProIle 420 425 430 Phe Asn Met Glu Asp Ile Glu Glu Gly Phe Glu Gly Tyr GluAsp Glu 435 440 445 Arg Ser Leu Leu Met Ser Gln Lys Ser Leu Glu Lys ArgPhe Gly Gln 450 455 460 Ser Pro Ile Phe Ile Ala Ser Thr Phe Met Thr GlnGly Gly Ile Pro 465 470 475 480 Pro Ser Thr Asn Pro Gly Ser Leu Leu LysGlu Ala Ile His Val Ile 485 490 495 Ser Cys Gly Tyr Glu Asp Lys Thr GluTrp Gly Lys Glu Ile Gly Trp 500 505 510 Ile Tyr Gly Ser Val Thr Glu AspIle Leu Thr Gly Phe Lys Met His 515 520 525 Ala Arg Gly Trp Ile Ser IleTyr Cys Met Pro Leu Arg Pro Cys Phe 530 535 540 Lys Gly Ser Ala Pro IleAsn Leu Ser Asp Arg Leu Asn Gln Val Leu 545 550 555 560 Arg Trp Ala LeuGly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro 565 570 575 Ile Trp TyrGly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala 580 585 590 Tyr IleAsn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Val Ala 595 600 605 TyrCys Val Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile 610 615 620Pro Ala Ile Ser Asn Tyr Ala Gly Ala Phe Phe Ile Leu Leu Phe Ala 625 630635 640 Ser Ile Phe Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly645 650 655 Ile Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly GlyThr 660 665 670 Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys ValLeu Ala 675 680 685 Gly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala ThrAsp Asp Asp 690 695 700 Gly Asp Phe Ala Glu Leu Tyr Val Phe Lys Trp ThrThr Leu Leu Ile 705 710 715 720 Pro Pro Thr Thr Val Leu Val Ile Asn LeuVal Gly Ile Val Ala Gly 725 730 735 Val Ser Tyr Ala Ile Asn Ser Gly TyrGln Ser Trp Gly Pro Leu Phe 740 745 750 Gly Lys Leu Phe Phe Ala Ile TrpVal Ile Leu His Leu Tyr Pro Phe 755 760 765 Leu Lys Gly Leu Met Gly LysGln Asn Arg Thr Pro Thr Ile Val Ile 770 775 780 Val Trp Ser Val Leu LeuAla Ser Ile Phe Ser Leu Leu Trp Val Lys 785 790 795 800 Ile Asp Pro PheIle Ser Pro Thr Gln Lys Ala Leu Ser Arg Gly Gln 805 810 815 Cys Gly ValAsn Cys 820 3 25 DNA Zea mays 3 cctctaagtc gcatagttcc gatat 25 4 25 DNAZea mays 4 tcagcagttt acaccacact gccca 25 5 3799 DNA Zea mays CDS(238)...(3477) misc_feature (1)...(3799) n = A,T,C or G 5 caactcacgttgccgcggct tcctccatcg gtgcggtgcc ctgtcctttt ctctcctcca 60 cctccctagtccctcctccc ccccgcatac atagctacta ctagtagcac cacgctcgca 120 gcgggagatgcggtgctgat ccgtgcccct gctcggatct cgggagtggt gccgacttgt 180 gtcgcttcggctctgcctag gccagctcct tgtcggttct gggcgagctc gcctgcc atg 240 Met 1 gagggc gac gcg gac ggc gtg aag tcg ggg agg cgc ggg gga ggg cag 288 Glu GlyAsp Ala Asp Gly Val Lys Ser Gly Arg Arg Gly Gly Gly Gln 5 10 15 gtg tgccag atc tgc ggc gat ggc gtg ggc act acg gcg gag gga gac 336 Val Cys GlnIle Cys Gly Asp Gly Val Gly Thr Thr Ala Glu Gly Asp 20 25 30 gtc ttc accgcc tgc gac gtc tgc ggg ttc ccg gtg tgc cgc ccc tgc 384 Val Phe Thr AlaCys Asp Val Cys Gly Phe Pro Val Cys Arg Pro Cys 35 40 45 tac gag tac gagcgc aag gac ggc aca caa gcg tgc ccc cag tgc aaa 432 Tyr Glu Tyr Glu ArgLys Asp Gly Thr Gln Ala Cys Pro Gln Cys Lys 50 55 60 65 aac aag tac aagcgc cac aag ggg agt cca gcg atc cga ggg gag gaa 480 Asn Lys Tyr Lys ArgHis Lys Gly Ser Pro Ala Ile Arg Gly Glu Glu 70 75 80 gga gac gat act gatgcc gat gat gct agc gac ttc aac tac cct gca 528 Gly Asp Asp Thr Asp AlaAsp Asp Ala Ser Asp Phe Asn Tyr Pro Ala 85 90 95 tct ggc aat gac gac cagaag cag aag att gct gac agg atg cgc agc 576 Ser Gly Asn Asp Asp Gln LysGln Lys Ile Ala Asp Arg Met Arg Ser 100 105 110 tgg cgc atg aat gct gggggc agc ggg gat gtt ggc cgc ccc aag tat 624 Trp Arg Met Asn Ala Gly GlySer Gly Asp Val Gly Arg Pro Lys Tyr 115 120 125 gac agt ggt gag atc gggctt acc aag tac gac agt ggt gag atc cct 672 Asp Ser Gly Glu Ile Gly LeuThr Lys Tyr Asp Ser Gly Glu Ile Pro 130 135 140 145 cgg gga tac atc ccgtca gtc act aac agc cag att tcg gga gaa atc 720 Arg Gly Tyr Ile Pro SerVal Thr Asn Ser Gln Ile Ser Gly Glu Ile 150 155 160 cct ggt gct tcc cctgac cat cat atg atg tct cct act ggg aac att 768 Pro Gly Ala Ser Pro AspHis His Met Met Ser Pro Thr Gly Asn Ile 165 170 175 ggc agg cgc gcc ccattt ccc tat atg aat cat tca tca aat ccg tcg 816 Gly Arg Arg Ala Pro PhePro Tyr Met Asn His Ser Ser Asn Pro Ser 180 185 190 agg gaa ttc tct ggtagc gtt ggg aat gtt gcc tgg aaa gag agg gtt 864 Arg Glu Phe Ser Gly SerVal Gly Asn Val Ala Trp Lys Glu Arg Val 195 200 205 gat ggc tgg aaa atgaag cag gac aag gga aca att ccc atg acg aat 912 Asp Gly Trp Lys Met LysGln Asp Lys Gly Thr Ile Pro Met Thr Asn 210 215 220 225 ggc aca agc attgct ccc tct gag ggc cgg ggt gtt ggt gat att gat 960 Gly Thr Ser Ile AlaPro Ser Glu Gly Arg Gly Val Gly Asp Ile Asp 230 235 240 gca tca act gattac aac atg gaa gat gcc tta tta aac gat gaa act 1008 Ala Ser Thr Asp TyrAsn Met Glu Asp Ala Leu Leu Asn Asp Glu Thr 245 250 255 cgc cag cct ctatct agg aaa gtt cca ctt cct tcc tcc agg ata aat 1056 Arg Gln Pro Leu SerArg Lys Val Pro Leu Pro Ser Ser Arg Ile Asn 260 265 270 cca tac agg atggtc att gtg cta cga ttg att gtt cta agc atc ttc 1104 Pro Tyr Arg Met ValIle Val Leu Arg Leu Ile Val Leu Ser Ile Phe 275 280 285 ttg cac tac cggatc aca aat cct gtg cgt aat gca tac cca ctg tgg 1152 Leu His Tyr Arg IleThr Asn Pro Val Arg Asn Ala Tyr Pro Leu Trp 290 295 300 305 ctt cta tctgtt ata tgt gag atc tgg ttt gct ctt tcc tgg ata ttg 1200 Leu Leu Ser ValIle Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu 310 315 320 gat cag tttcca aag tgg ttt cca atc aac cgc gag act tac ctt gat 1248 Asp Gln Phe ProLys Trp Phe Pro Ile Asn Arg Glu Thr Tyr Leu Asp 325 330 335 aga ctc gcatta agg tat gac cgg gaa ggt gag cca tct cag ttg gct 1296 Arg Leu Ala LeuArg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala 340 345 350 gct gtt gacatt ttt gtc agt act gtc gac cca atg aag gag cct cct 1344 Ala Val Asp IlePhe Val Ser Thr Val Asp Pro Met Lys Glu Pro Pro 355 360 365 ctt gtc actgcc aat acc gtg cta tcc att ctc gct gtg gac tat cct 1392 Leu Val Thr AlaAsn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro 370 375 380 385 gtg gataag gtc tct tgc tat gta tct gat gat gga gct gct atg ctg 1440 Val Asp LysVal Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu 390 395 400 aca tttgat gca cta gct gag act tca gag ttt gct aga aaa tgg gtg 1488 Thr Phe AspAla Leu Ala Glu Thr Ser Glu Phe Ala Arg Lys Trp Val 405 410 415 cca tttgtt aag aag tac aac att gaa cct aga gct cct gaa tgg tac 1536 Pro Phe ValLys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr 420 425 430 ttc tcccag aaa att gat tac ttg aag gac aaa gtg cac cct tca ttt 1584 Phe Ser GlnLys Ile Asp Tyr Leu Lys Asp Lys Val His Pro Ser Phe 435 440 445 gtt aaagac cgc cgg gcc atg aag aga gaa tat gaa gaa ttc aaa att 1632 Val Lys AspArg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile 450 455 460 465 agggta aat ggc ctt gtt gct aag gca caa aaa gtc cct gag gaa gga 1680 Arg ValAsn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly 470 475 480 tggatc atg caa gat ggc aca cca tgg cca gga aac aat acc agg gac 1728 Trp IleMet Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp 485 490 495 catcct gga atg att cag gtt ttc ctt ggt cac agt ggt ggt ctt gat 1776 His ProGly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp 500 505 510 actgag ggt aat gag cta ccc cgt ttg gtc tat gtt tct cgt gaa aaa 1824 Thr GluGly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys 515 520 525 cgtcct gga ttc cag cat cac aag aaa gct ggt gcc atg aat gct ctt 1872 Arg ProGly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu 530 535 540 545gtc cgc gtc tca gct gtg ctt acc aat gga caa tac atg ttg aat ctt 1920 ValArg Val Ser Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn Leu 550 555 560gat tgt gat cac tac atc aac aac agt aag gct ctc agg gaa gct atg 1968 AspCys Asp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met 565 570 575tgc ttc ctt atg gat cct aac cta gga agg agt gtc tgc tat gtt cag 2016 CysPhe Leu Met Asp Pro Asn Leu Gly Arg Ser Val Cys Tyr Val Gln 580 585 590ttt ccc cag agg ttc gat ggt att gat agg aat gat cga tat gcc aac 2064 PhePro Gln Arg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn 595 600 605agg aac acc gtg ttt ttc gat att aac ttg aga ggt ctt gat ggc atc 2112 ArgAsn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile 610 615 620625 caa gga cca gtt tat gtg ggc act ggc tgt gtt ttc aac aga aca gct 2160Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala 630 635640 cta tat ggt tat gag ccc cca att aag caa aag aag ggt ggt ttc ttg 2208Leu Tyr Gly Tyr Glu Pro Pro Ile Lys Gln Lys Lys Gly Gly Phe Leu 645 650655 tca tca cta tgt ggt ggc agg aag aag gga agc aaa tca aag aag ggc 2256Ser Ser Leu Cys Gly Gly Arg Lys Lys Gly Ser Lys Ser Lys Lys Gly 660 665670 tca gac aag aaa aag tca cag aag cat gtg gac agt tct gtg cca gta 2304Ser Asp Lys Lys Lys Ser Gln Lys His Val Asp Ser Ser Val Pro Val 675 680685 ttc aat ctt gaa gat ata gag gag gga gtt gaa ggc gct gga ttt gat 2352Phe Asn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly Ala Gly Phe Asp 690 695700 705 gat gag aaa tca ctt ctt atg tct caa atg agc ttg gag aag aga ttt2400 Asp Glu Lys Ser Leu Leu Met Ser Gln Met Ser Leu Glu Lys Arg Phe 710715 720 ggc caa tct gca gct ttt gtt gcg tcc act ctg atg gaa tat ggt ggt2448 Gly Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly 725730 735 gtt cct cag tct gcg act cca gaa tct ctt ctg aaa gaa gct atc cat2496 Val Pro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His 740745 750 gtc ata agt tgt ggc tac gag gac aag att gaa tgg gga act gag att2544 Val Ile Ser Cys Gly Tyr Glu Asp Lys Ile Glu Trp Gly Thr Glu Ile 755760 765 ggg tgg atc tat ggt tct gtg acg gaa gat att ctc act ggg ttc aag2592 Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys 770775 780 785 atg cac gca cga ggc tgg cgg tcg atc tac tgc atg cct aag cggccg 2640 Met His Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro790 795 800 gcc ttc aag gga tcg gct ccc atc aat ctc tca gac cgt ctg aaccag 2688 Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln805 810 815 gtg ctc cgg tgg gct ctc ggt tca gtg gaa atc ctt ttc agc cggcat 2736 Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Phe Ser Arg His820 825 830 tgc ccc cta tgg tac ggg tac gga gga cgc ctg aag ttc ttg gagaga 2784 Cys Pro Leu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu Arg835 840 845 ttc gcc tac atc aac acc acc atc tac ccg ctc acg tcc ctc ccgctc 2832 Phe Ala Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Leu Pro Leu850 855 860 865 ctc att tac tgt atc ctg cct gcc atc tgc ctg ctc acg gggaag ttc 2880 Leu Ile Tyr Cys Ile Leu Pro Ala Ile Cys Leu Leu Thr Gly LysPhe 870 875 880 atc atc cca gag atc agc aac ttc gct agt atc tgg ttc atctct ctc 2928 Ile Ile Pro Glu Ile Ser Asn Phe Ala Ser Ile Trp Phe Ile SerLeu 885 890 895 ttc atc tcg atc ttc gcc acg ggt atc ctg gag atg agg tggagc ggc 2976 Phe Ile Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp SerGly 900 905 910 gtg ggc atc gac gag tgg tgg agg aac gag cag ttc tgg gtcatc gga 3024 Val Gly Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val IleGly 915 920 925 ggc atc tcc gcc cac ctc ttc gcc gtc ttc cag ggc ctc ctcaag gtg 3072 Gly Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu LysVal 930 935 940 945 ctt gcc ggc atc gac acc aac ttc acc gtc acc tcc aaggcc tcg gat 3120 Leu Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys AlaSer Asp 950 955 960 gaa gac ggc gac ttc gcg gag ctg tac atg ttc aag tggacg aca ctt 3168 Glu Asp Gly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp ThrThr Leu 965 970 975 ctg atc ccg ccc acc acc atc ctg atc atc aac ctg gtcggc gtt gtt 3216 Leu Ile Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val GlyVal Val 980 985 990 gcc ggc atc tcc tac gcc atc aac agc ggg tac cag tcgtgg ggt ccg 3264 Ala Gly Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser TrpGly Pro 995 1000 1005 ctc ttc ggc aag ctc ttc ttc gcc ttc tgg gtg atcgtt cac ctg tac 3312 Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile ValHis Leu Tyr 1010 1015 1020 1025 ccg ttc ctc aag ggt ctc atg ggt cgg cagaac cgc acc ccg acc atc 3360 Pro Phe Leu Lys Gly Leu Met Gly Arg Gln AsnArg Thr Pro Thr Ile 1030 1035 1040 gtg gtt gtc tgg gcg atc ctg ctg gcgtcg atc ttc tcc ttg ctg tgg 3408 Val Val Val Trp Ala Ile Leu Leu Ala SerIle Phe Ser Leu Leu Trp 1045 1050 1055 gtt cgc atc gat ccg ttc acc aaccgc gtc act ggc ccg gat act cga 3456 Val Arg Ile Asp Pro Phe Thr Asn ArgVal Thr Gly Pro Asp Thr Arg 1060 1065 1070 acg tgt ggc atc aac tgc tagggaggtggaa ggtttgtaga aacagagaga 3507 Thr Cys Gly Ile Asn Cys * 1075taccacgaat gtgccgctgc cacaaattgt ctgttagtaa gttatatagg caggtggcgt 3567tatttacagc tacgtacaca caaggggata ctccgtttat cactggtgtg cattcttttg 3627ttgatataag ttactatata tacgtattgc ttctactttg tggagagtgg ctgacaggac 3687cagttttgta atgttatgaa cagcaaagaa ataagttagt ttccaaaaaa aaaaaaaaaa 3747aaaaaaaaan aaaaaaaaaa aaaaaaanan aaaanaaaaa aaaaaaaacc cc 3799 6 1079PRT Zea mays 6 Met Glu Gly Asp Ala Asp Gly Val Lys Ser Gly Arg Arg GlyGly Gly 1 5 10 15 Gln Val Cys Gln Ile Cys Gly Asp Gly Val Gly Thr ThrAla Glu Gly 20 25 30 Asp Val Phe Thr Ala Cys Asp Val Cys Gly Phe Pro ValCys Arg Pro 35 40 45 Cys Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala CysPro Gln Cys 50 55 60 Lys Asn Lys Tyr Lys Arg His Lys Gly Ser Pro Ala IleArg Gly Glu 65 70 75 80 Glu Gly Asp Asp Thr Asp Ala Asp Asp Ala Ser AspPhe Asn Tyr Pro 85 90 95 Ala Ser Gly Asn Asp Asp Gln Lys Gln Lys Ile AlaAsp Arg Met Arg 100 105 110 Ser Trp Arg Met Asn Ala Gly Gly Ser Gly AspVal Gly Arg Pro Lys 115 120 125 Tyr Asp Ser Gly Glu Ile Gly Leu Thr LysTyr Asp Ser Gly Glu Ile 130 135 140 Pro Arg Gly Tyr Ile Pro Ser Val ThrAsn Ser Gln Ile Ser Gly Glu 145 150 155 160 Ile Pro Gly Ala Ser Pro AspHis His Met Met Ser Pro Thr Gly Asn 165 170 175 Ile Gly Arg Arg Ala ProPhe Pro Tyr Met Asn His Ser Ser Asn Pro 180 185 190 Ser Arg Glu Phe SerGly Ser Val Gly Asn Val Ala Trp Lys Glu Arg 195 200 205 Val Asp Gly TrpLys Met Lys Gln Asp Lys Gly Thr Ile Pro Met Thr 210 215 220 Asn Gly ThrSer Ile Ala Pro Ser Glu Gly Arg Gly Val Gly Asp Ile 225 230 235 240 AspAla Ser Thr Asp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu 245 250 255Thr Arg Gln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser Ser Arg Ile 260 265270 Asn Pro Tyr Arg Met Val Ile Val Leu Arg Leu Ile Val Leu Ser Ile 275280 285 Phe Leu His Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala Tyr Pro Leu290 295 300 Trp Leu Leu Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser TrpIle 305 310 315 320 Leu Asp Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg GluThr Tyr Leu 325 330 335 Asp Arg Leu Ala Leu Arg Tyr Asp Arg Glu Gly GluPro Ser Gln Leu 340 345 350 Ala Ala Val Asp Ile Phe Val Ser Thr Val AspPro Met Lys Glu Pro 355 360 365 Pro Leu Val Thr Ala Asn Thr Val Leu SerIle Leu Ala Val Asp Tyr 370 375 380 Pro Val Asp Lys Val Ser Cys Tyr ValSer Asp Asp Gly Ala Ala Met 385 390 395 400 Leu Thr Phe Asp Ala Leu AlaGlu Thr Ser Glu Phe Ala Arg Lys Trp 405 410 415 Val Pro Phe Val Lys LysTyr Asn Ile Glu Pro Arg Ala Pro Glu Trp 420 425 430 Tyr Phe Ser Gln LysIle Asp Tyr Leu Lys Asp Lys Val His Pro Ser 435 440 445 Phe Val Lys AspArg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys 450 455 460 Ile Arg ValAsn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu 465 470 475 480 GlyTrp Ile Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg 485 490 495Asp His Pro Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu 500 505510 Asp Thr Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu 515520 525 Lys Arg Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala530 535 540 Leu Val Arg Val Ser Ala Val Leu Thr Asn Gly Gln Tyr Met LeuAsn 545 550 555 560 Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala LeuArg Glu Ala 565 570 575 Met Cys Phe Leu Met Asp Pro Asn Leu Gly Arg SerVal Cys Tyr Val 580 585 590 Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp ArgAsn Asp Arg Tyr Ala 595 600 605 Asn Arg Asn Thr Val Phe Phe Asp Ile AsnLeu Arg Gly Leu Asp Gly 610 615 620 Ile Gln Gly Pro Val Tyr Val Gly ThrGly Cys Val Phe Asn Arg Thr 625 630 635 640 Ala Leu Tyr Gly Tyr Glu ProPro Ile Lys Gln Lys Lys Gly Gly Phe 645 650 655 Leu Ser Ser Leu Cys GlyGly Arg Lys Lys Gly Ser Lys Ser Lys Lys 660 665 670 Gly Ser Asp Lys LysLys Ser Gln Lys His Val Asp Ser Ser Val Pro 675 680 685 Val Phe Asn LeuGlu Asp Ile Glu Glu Gly Val Glu Gly Ala Gly Phe 690 695 700 Asp Asp GluLys Ser Leu Leu Met Ser Gln Met Ser Leu Glu Lys Arg 705 710 715 720 PheGly Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met Glu Tyr Gly 725 730 735Gly Val Pro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile 740 745750 His Val Ile Ser Cys Gly Tyr Glu Asp Lys Ile Glu Trp Gly Thr Glu 755760 765 Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe770 775 780 Lys Met His Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro LysArg 785 790 795 800 Pro Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser AspArg Leu Asn 805 810 815 Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu IleLeu Phe Ser Arg 820 825 830 His Cys Pro Leu Trp Tyr Gly Tyr Gly Gly ArgLeu Lys Phe Leu Glu 835 840 845 Arg Phe Ala Tyr Ile Asn Thr Thr Ile TyrPro Leu Thr Ser Leu Pro 850 855 860 Leu Leu Ile Tyr Cys Ile Leu Pro AlaIle Cys Leu Leu Thr Gly Lys 865 870 875 880 Phe Ile Ile Pro Glu Ile SerAsn Phe Ala Ser Ile Trp Phe Ile Ser 885 890 895 Leu Phe Ile Ser Ile PheAla Thr Gly Ile Leu Glu Met Arg Trp Ser 900 905 910 Gly Val Gly Ile AspGlu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile 915 920 925 Gly Gly Ile SerAla His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys 930 935 940 Val Leu AlaGly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser 945 950 955 960 AspGlu Asp Gly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr 965 970 975Leu Leu Ile Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val 980 985990 Val Ala Gly Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly 9951000 1005 Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val HisLeu 1010 1015 1020 Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn ArgThr Pro Thr 1025 1030 1035 1040 Ile Val Val Val Trp Ala Ile Leu Leu AlaSer Ile Phe Ser Leu Leu 1045 1050 1055 Trp Val Arg Ile Asp Pro Phe ThrAsn Arg Val Thr Gly Pro Asp Thr 1060 1065 1070 Arg Thr Cys Gly Ile AsnCys 1075 7 25 DNA Zea mays 7 atggagggcg acgcggacgg cgtga 25 8 25 DNA Zeamays 8 ctagcagttg atgccacacg ttcga 25 9 36 DNA Artificial SequenceDesigned oligonucleotide based upon the adapter sequence and poly T toremove clones which have a poly A tail but no cDNA. 9 tcgacccacgcgtccgaaaa aaaaaaaaaa aaaaaa 36 10 27 DNA Zea mays 10 tgctgatatcgagaaggccg gaatcgt 27 11 21 DNA Zea mays 11 ctccccacca gacccttgag g 2112 32 DNA Zea mays 12 agagaagcca acgccawcgc ctcyatttcg tc 32 13 36 DNAArtificial Sequence removes clones containing a poly A tail but no cDNA13 tcgacccacg cgtccgaaaa aaaaaaaaaa aaaaaa 36 14 27 DNA Zea mays 14tcgtgatatc gagaaggccg gaatcgt 27 15 21 DNA Zea mays 15 ctccccaccagacccttgag g 21 16 32 DNA Zea mays 16 agagaagcca acgccawcgc ctcyatttcgtc 32 17 9 DNA Zea mays 17 tcttcacca 9 18 9 DNA Zea mays 18 ggtccttcg 919 9 DNA Zea mays 19 gtcgaaatt 9 20 9 DNA Zea mays 20 ttcttcagc 9 21 9DNA Zea mays 21 gctcacggg 9 22 9 DNA Zea mays 22 gaagtttat 9

What is claimed is:
 1. An isolated nucleic acid comprising a memberselected from the group consisting of: (a) a polynucleotide having atleast 80% sequence identity, as determined by the GAP algorithm underdefault parameters, to a polynucleotide selected from the groupconsisting of SEQ ID NOS: 1 and 5; (b) a polynucleotide encoding apolypeptide selected from the group consisting of SEQ ID NOS: 2 and 6;(c) a polynucleotide amplified from a Zea mays nucleic acid libraryusing primers which selectively hybridize, under stringent hybridizationconditions, to loci within a polynucleotide selected from the groupconsisting of SEQ ID NOS: 1 and 5; (d) a polynucleotide whichselectively hybridizes, under stringent hybridization conditions and awash in 0.1×X SSC at 65° C., to a polynucleotide selected from the groupconsisting of SEQ ID NOS: 1 and 5; (e) a polynucleotide selected fromthe group consisting of SEQ ID NOS: 1 and 5; (f) a polynucleotide whichis complementary to a polynucleotide of (a), (b), (c), (d), or (e); and(g) a polynucleotide comprising at least 25 contiguous nucleotides froma polynucleotide of (a), (b), (c), (d), (e), or (f).
 2. A recombinantexpression cassette, comprising a member of claim 1 operably linked, insense or anti-sense orientation, to a promoter.
 3. A host cellcomprising the recombinant expression cassette of claim
 2. 4. Atransgenic plant comprising the recombinant expression cassette of claim2.
 5. The transgenic plant of claim 4, wherein said plant is a monocot.6. The transgenic plant of claim 4, wherein said plant is a dicot. 7.The transgenic plant of claim 4, wherein said plant is selected from thegroup consisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, peanut, and cocoa.
 8. A seed fromthe transgenic plant of claim
 4. 9. A method of modulating the level ofcellulose synthase in a plant cell, comprising: (a) introducing into aplant cell a recombinant expression cassette comprising a polynucleotideof claim 1 operably linked to a promoter; (b) culturing the plant cellunder plant cell growing conditions; and (c) inducing expression of saidpolynucleotide for a time sufficient to modulate the level of cellulosesynthase in said plant cell.
 10. The method of claim 9, wherein theplant cell is from maize, wheat, rice, or soybean.
 11. A method ofmodulating the level of cellulose synthase in a plant, comprising: (a)introducing into a plant cell a recombinant expression cassettecomprising a polynucleotide of claim 1 operably linked to a promoter;(b) culturing the plant cell under plant cell growing conditions; (c)regenerating a plant from said plant cell; and (d) inducing expressionof said polynucleotide for a time sufficient to modulate the level ofcellulose synthase in said plant.
 12. The method of claim 11, whereinthe plant is maize, wheat, rice, or soybean.
 13. An isolated proteincomprising a member selected from the group consisting of: (a) apolypeptide of at least 20 contiguous amino acids from a polypeptideselected from the group consisting of SEQ ID NOS: 2 and 6; (b) apolypeptide selected from the group consisting of SEQ ID NOS: 2 and 6;(c) a polypeptide having at least 80% sequence identity to, and havingat least one epitope in common with, a polypeptide selected from thegroup consisting of SEQ ID NOS: 2 and 6, wherein said sequence identityis determined by the GAP algorithm under default parameters; and, (d) atleast one polypeptide encoded by a member of claim
 1. 14. A method ofmodifying expression of a cellulose synthase gene in a maize plant,comprising: (a) identifying, from a population of maize plantsmutagenized with the Mu transposable element, those plants containingone or more Mu insertions within a polynucleotide of claim 1; (b)selecting those plants showing modified cellulose synthase geneexpression.
 15. The method of claim 14, where expression of thecellulose synthase gene is down-regulated.